Donald G. MacPhee

Colorectal Cancer With Mutation in BRAF, KRAS, and Wild-Type With Respect to Both Oncogenes Showing Different Patterns of DNA Methylation

PURPOSE BRAF mutations are common in sporadic colorectal cancers (CRCs) with a DNA mismatch repair (MMR) deficiency that results from promoter methylation of hMLH1, whereas KRAS mutations are common in MMR proficient CRCs associated with promoter methylation of MGMT. The aim of this study was to further investigate the link between genetic alterations in the RAS/RAF/ERK pathway and an underlying epigenetic disorder. PATIENTS AND METHODS Activating mutations of BRAF and KRAS were identified and correlated with promoter methylation of 11 loci, including MINT1, MINT2, MINT31, CACNA1G, p16(INK4a), p14(ARF), COX2, DAPK, MGMT, and the two regions in hMLH1 in 468 CRCs and matched normal mucosa. RESULTS BRAF V599E mutations were identified in 21 (9%) of 234 CRCs, and KRAS mutations were identified in 72 (31%) of 234 CRCs. Mutations in BRAF and KRAS were never found in the same tumor. CRCs with BRAF mutations showed high-level promoter methylation in multiple loci, with a mean number of methylated loci of 7.2 (95% CI, 6.6 to 7.9) among 11 loci examined (P < .0001). Tumors with KRAS mutations showed low-level promoter methylation, and CRCs with neither mutation showed a weak association with promoter methylation, with an average number of methylated loci of 1.8 (95% CI, 1.5 to 2.1) and 1.0 (95% CI, 0.79 to 1.3), respectively. CONCLUSION In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.

Radiation dose-dependent increases in inflammatory response markers in A-bomb survivors

Purpose : The well-documented increases in malignant tumours in the A-bomb survivors have recently been supplemented by reports that non-cancer diseases, including cardiovascular disease, may also have increased in incidence with increasing radiation dose. Given that low-level inflammatory responses are widely accepted as a significant risk factor for such diseases, we undertook a detailed investigation of the long-term effects of ionizing radiation on the levels of the inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6) in A-bomb survivors. Materials and methods : Blood samples were taken from 453 participants in a long-term epidemiological cohort of A-bomb survivors. Plasma levels of CRP and IL-6 were measured using standard antibody-mediated procedures. Relationships between CRP or IL-6 levels and radiation dose were then investigated by multivariate regression analysis. Blood lymphocytes from each individual were used for immunophenotyping by flow cytometry with murine monoclonal antibodies to CD3, CD4 and CD8. Results : CRP levels were significantly increased by about 31% Gy −1 of estimated A-bomb radiation (p =0.0001). Higher CRP levels also correlated with age, male gender, body mass index and a history of myocardial infarction. After adjustments for these factors, CRP levels still appeared to have increased significantly with increasing radiation dose (about 28% increase at 1Gy, p =0.0002). IL-6 levels also appeared to have increased with radiation dose by 9.3% at 1Gy (p =0.0003) and after multiple adjustments by 9.8% at 1Gy (p =0.0007). The elevated CRP and IL-6 levels were associated with decreases in the percentages of CD4 + helper T-cells in peripheral blood lymphocyte populations. Conclusions : Our results appear to indicate that exposure to A-bomb radiation has caused significant increases in inflammatory activity that are still demonstrable in the blood of A-bomb survivors and which may lead to increased risks of cardiovascular disease and other non-cancer diseases.

Effect of rec mutations on the ultraviolet protecting and mutation-enhancing properties of the plasmid R-Utrecht in Salmonella typhimurium.

Derivatives of strains of S. typhimurium with and without H-Utrecht were tested for their ability to yield uv-induced back mutations of the amber mutation. Results showed that R-Utrecht significantly enhanced the uv mutability of one strain, but cells of another strain were stable to the mutagenic effects of uv whether or not R-Utrecht is present. It was concluded that because the R-Utrecht factor does not restore uv resistance or uv mutability to the recA-type mutants, it seems unlikely that the effects of the R factor on the uv response of wild- type cells could be due to a plasmid gene whose product is functionally equivalent to that of the chromosomal recA. (HLW)

Spontaneous, ultraviolet and ionizing radiation mutagenesis in two auxotrophic strains of Salmonella typhimurium carrying an R plasmid

Ultraviolet-induced, gamma-induced and spontaneous mutation yields were studied in two different auxotrophic strains of Salmonella typhimurium in the presence and absence of the UV-protecting drug resistance transfer factor R-Utrecht. One strain, carrying the hisC527 (amber) mutation, showed significantly increased spontaneous, UV- and gamma-induced mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R factor, but appeared to show no significant increase in spontaneous mutability and only a very slight increase in gamma-mutability when carrying the R factor. These results demonstrate that the R-Utrecht plasmid, known to enhance UV-induced mutation yields in S. typhimurium, can also significantly enhance both spontaneous and gamma-induced mutation yields in this species. The latter effects are not so discernible with all markers, however, as shown by the results with strains carrying the trpD1 mutation. Enhancement of spontaneous mutability thus appears to be correlated with enhancement of gamma-mutability rather than UV mutability.

The role of DNA polymerase I and the rec system in survival of bacteria and bacteriophages damaged by the photodynamic action of acridine orange

SummaryThe effect of acridine orange (AO)-sensitized photodynamic treatment (PD) was studied in various repair-deficient mutants of Salmonella typhimurium and Escherichia coli. Bacteria of either species carrying mutations in the polA gene and hence deficient in the enzyme DNA polymerase I were significantly more sensitive to PD-killing than polA+ parent bacteria or phenotypically POL+ revertants of the polA strains (selected on the basis of resistance to methyl methanesulphonate). It therefore appears that DNA polymerase I plays an important role in cellular recovery from PD treatment. E. coli carrying a mutation in the recA gene was also more sensitive to PD-treatment than its parent strain, as was S. typhimurium carrying a mutation of the recA type. In S. typhimurium the rec mutant was somewhat less sensitive to PD-killing than the pol mutant even although it is much more sensitive to ultraviolet killing. E. coli strains with mutations in the recB and recC genes were intermediate in PD sensitivity between the recA and the parent strain. S. typhimurium and E. coli bacteria with mutations in the polA and recA genes showed reduced ability to host-cell reactivate PD-damaged bacteriophages ES 18 and λc1, indicating that the polA+ and recA+ gene products also contribute to repair of bacteriophages damaged by PD treatment. It is suggested that the recombinational repair process is less important for recovery from PD than for recovery from UV, and that the primary contribution of the rec genes to recovery from PD may be in repair of single-strand gaps by repair resynthesis.

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.

Comparative studies of mutagenic, DNA binding and antileukaemic properties of 9-anilinoacridine derivatives and related compounds

Abstract Many derivatives of 9-anilinoacridine show significant cytotoxic activity against mouse L1210 leukaemia cells in vitro. We have compared the mutagenic activity of three of these compounds with those of (i) ICR191, an acridine half-mustard which also inhibits the growth of mouse L1210 cells in vitro and (ii) 9-aminoacridine, which is significantly less cytotoxic than ICR191 towards mouse L1210 cells. Induced mutations have been measured in derivatives of the Salmonella typhimurium frameshift strain hisC3076, differing in DNA repair capabilities. The anilinoacridines chosen included the clinical antitumou drug 4′-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) and two closely related derivatives, one active and one inactive towards experimental leukaemia in mice. The patterns of mutagenesis observed for the three anilinoacridine derivatives were found to resemble those of 9-aminoacridine rather than those of ICR191, except that mutagenesis by 9-aminoacridine appeared to be recA-independent, whereas that by the anilinoacridines and ICR191 appeared to be at least partially dependent upon the recA+ gene product. As with 9-aminoacridine, mutagenesis by all three 9-anilinoacridine derivatives was not detectably enhanced by the N group plasmid pKM101 (known to enhance many other kinds of chemical and radiation mutation yields) nor was it enhanced by absence of the cellular excision-repair system (i.e. in a uvrB deletion mutant). By contrast, mutagenesis by ICR191 was significantly enhanced by the N group plasmid pKM101 and was also enhanced in the uvrB strain. The effects of two different polA alleles on mutagenesis by 9-aminoacridine and the AMSA compounds were also noted; interestingly, while both the polA1 and polA3 alleles significantly reduced mutagenesis by all four of these compounds, the plasmid pKM101 restored mutagenesis almost to wild type levels in the polA1 background but had no such major effect in the polA3 background. The effect of the polA1 allele on mutagenesis by ICR191 was the same as for the AMSA compounds, while the polA3 allele increased rather than decreased frameshift mutagenesis by this compound.

Induction of base-pair substitution and prameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD30529 We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-INDUCED BASE-PAIR SUBSTITUTION MUTATIONS ARIse by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: Does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

Abstract Kusunoki, Y., Yamaoka, M., Kasagi, F., Hayashi, T., Koyama, K., Kodama, K., MacPhee, D. G. and Kyoizumi, S. T Cells of Atomic Bomb Survivors Respond Poorly to Stimulation by Staphylococcus aureus Toxins In Vitro: Does This Stem from their Peripheral Lymphocyte Populations Having a Diminished Naïve CD4 T-Cell Content? Radiat. Res. 158, 715–724 (2002). We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer naïve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (naïve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (naïve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their naïve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.

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.