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S M Galloway

Publications and source records attributed to S M Galloway.

At least 19 recordsLinked to original sources
PubMed2002

Bmp15 mutations and ovarian function.

BMP15, also known as growth and differentiation factor 9B (GDF9B), is a member of the transforming growth factor beta superfamily (TGFbeta) which in humans, rodents and sheep is expressed exclusively in the oocyte. BMP15 is closely related to GDF9, another oocyte-specific member of this superfamily which has been shown to be essential for early ovarian folliculogenesis. Inactivation of the BMP15 gene in mice has shown only minor effects on fertility. However, Inverdale and Hanna lines of sheep carry naturally occurring mutations in BMP15 which highlight differences in the action of this gene between mice and other mammals. Sheep which are heterozygous show an increase in ovulation rate whereas homozygotes are infertile. The granulosa cell receptor which mediates the BMP15 response has not yet been identified, but the discovery that a point mutation in the BMP1B receptor in Booroola sheep is responsible for increased ovulation rate highlights the importance of the TGFbeta signalling molecules in early folliculogenesis.

Animals↗
PubMed2001

Genetic mutations influencing ovulation rate in sheep.

Ovulation rate in mammals is determined by a complex exchange of endocrine signals between the pituitary gland and the ovary, and by paracrine signals within ovarian follicles between the oocyte and its adjacent somatic cells. One approach to identifying factors regulating ovulation rate is to find mutations that influence the target phenotype and, in this context, sheep are proving to be remarkable experimental models. Recently, in three sheep families, namely Inverdale, Hanna and Booroola, the inherited mutation was mapped to a specific region of the sheep X chromosome (Inverdale, Hanna) or sheep chromosome 6 (Booroola) and in each, a point mutation was identified in genes from the bone morphogenetic protein (BMP) relatives of the transforming growth factor beta superfamily or their receptors. In Inverdale (I) and Hanna (H) sheep, separate point mutations were identified in the BMP15 gene corresponding to sites in the mature peptide coding region of the BMP15 growth factor (also known as growth differentiation factor 9B; GDF9B). Expression of the BMP15 gene was located exclusively in oocytes from the primary stage of follicular growth. There is a complete block of normal follicular development in females carrying two copies of the Inverdale mutation (II), two copies of the Hanna mutation (HH), or one copy of each mutation (HI). Increased ovulation rates are found in females with only one copy of either mutation (I+ or H+). In Booroola sheep, a point mutation was identified in the highly conserved intracellular serine threonine kinase signalling domain of the BMP-1B receptor. Within the ovary, this gene is expressed in oocytes in primordial and pre-antral follicles and in granulosa cells from the primary stage of growth as well as in corpora lutea. The effect of the Booroola mutation is additive for ovulation rate: animals with one copy of the mutation have an ovulation rate of 3 or 4, whereas those with two copies have an ovulation rate of between 5 and 14. Physiological studies of the above mutations demonstrate that the oocyte plays an active role with respect to its adjacent somatic cells during follicular development and support the hypothesis that the oocyte has a significant influence on the number of follicles that proceed to ovulation.

Animals↗
PubMed2001

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Animals↗
PubMed2001

Genes controlling ovulation rate in sheep.

Sheep provide a valuable model for studying the genetic control of ovulation rate. Recent progress includes the identification of mutations in BMP15 (bone morphogenetic protein 15) that increase ovulation rate in heterozygous carriers and block follicular development in homozygous carriers. The genes characterized to date appear to act principally within the ovary and result in earlier maturity of granulosa cells and reduced follicular size. There may also be other sites of action, and increased FSH concentrations appear to be important in the expression of the FecB phenotype. A new locus on the X chromosome in New Zealand Coopworth sheep increases ovulation rate by about 0.4 and is maternally imprinted. Results from studies in the Cambridge and Belclare breeds indicate that further genes remain to be characterized. Finding the first mutations leading directly to variation in ovulation rate is likely to speed up the identification and molecular analysis of these other genes. There is still much to learn about follicular development and the control of litter size from genetic models in sheep.

Animals↗
PubMed2000

Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies.

Phenolphthalein induces tumors in rodents but because it is negative in assays for mutation in Salmonella and in mammalian cells, for DNA adducts and for DNA strand breaks, its primary mechanism does not seem to be DNA damage. Chromosome aberration (Ab) induction by phenolphthalein in vitro is associated with marked cytotoxicity. At very high doses, phenolphthalein induces weak increases in micronuclei (MN) in mouse bone marrow; a larger response is seen with chronic treatment. All this suggests genotoxicity is a secondary effect that may not occur at lower doses. In heterozygous TSG-p53((R)) mice, phenolphthalein induces lymphomas and also MN, many with kinetochores (K), implying chromosome loss. Induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas. Here we address some of the postulated mechanisms of genotoxicity in vitro, including metabolic activation, inhibition of thymidylate synthetase, cytotoxicity, oxidative stress, DNA damage and aneuploidy. We show clearly that phenolphthalein does not require metabolic activation by S9 to induce Abs. Inhibition of thymidylate synthetase is an unlikely mechanism, since thymidine did not prevent Ab induction by phenolphthalein. Phenolphthalein dramatically inhibited DNA synthesis, in common with many non-DNA reactive chemicals that induce Abs at cytotoxic doses. Phenolphthalein strongly enhances levels of intracellular oxygen radicals (ROS). The radical scavenger DMSO suppresses phenolphthalein-induced toxicity and Abs whereas H(2)O(2) potentiates them, suggesting a role for peroxidative activation. Phenolphthalein did not produce DNA strand breaks in rat hepatocytes or DNA adducts in Chinese hamster ovary (CHO) cells. All the evidence points to an indirect mechanism for Abs that is unlikely to operate at low doses of phenolphthalein. We also found that phenolphthalein induces mitotic abnormalities and MN with kinetochores in vitro. These are also enhanced by H(2)O(2) and suppressed by DMSO. Our findings suggest that induction of Abs in vitro is a high-dose effect in oxidatively stressed cells and may thus have a threshold. There may be more than one mechanism operating in vitro and in vivo, possibly indirect genotoxicity at high doses and also chromosome loss, both of which would likely have a threshold.

Animals↗
PubMed2000

The genetic toxicity of the peroxisome proliferator class of rodent hepatocarcinogen.

Peroxisome proliferators comprise a structurally diverse class of chemicals. Some of the members of this class show evidence of genetic toxicity (most evidently the in vitro clastogen Wyeth 14,643, WY), while others do not (most evidently methyl clofenapate, MCP). When attempting to understand the mechanism of rodent hepatocarcinogenesis of this class of chemicals the possible role of genetic toxicity should be assessed on a class-wide basis, i.e., if just one peroxisome proliferator is shown to be unequivocally inactive as a genetic toxin, genetic toxicity cannot be implicated in the carcinogenic activity of peroxisome proliferators as a class. In an earlier paper, we established MCP as inactive in a range of in vitro and in vivo genetic toxicity assays. However, the top dose level of MCP that could be tested for induction of chromosome aberrations (clastogenicity) in human lymphocytes and CHO cells was limited by the relative insolubility of the test agent in the assay medium. Methyl clofenapate was not toxic up to a dose that produced precipitate, so cannot be directly compared with WY, which induced aberrations only at toxic dose levels. In the present paper, we have evaluated the clastogenicity of the carcinogenic peroxisome proliferator nafenopin (NAF) at dose levels up to those that are toxic to CHO cells, and found no evidence of chromosome aberration induction. These data isolate further the genetic toxicity of WY from other peroxisome proliferators, and increase confidence in the proposal that genetic toxicity does not play a critical role in the hepatocarcinogenicity of peroxisome proliferators.

Animals↗
PubMed2000

Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster.

Fas-mediated apoptosis is an important regulator of cell survival, and abnormalities in this system have been shown to result in a number of human pathological conditions. A secreted member of the tumor necrosis factor receptor superfamily, DcR3, was recently reported to be amplified in human lung and colon cancers as a negative regulator of Fas-mediated apoptosis. We identified this gene, which we call M68. M68 genomic DNA, mRNA, and protein levels were examined in a series of human gastrointestinal tract tumors. Using M68 immunohistochemistry and a scoring system similar to that used for HER-2/neu, we found that M68 protein was overexpressed in 30 of 68 (44%) human adenocarcinomas of the esophagus, stomach, colon, and rectum. Tumors examined by Northern blot revealed M68 mRNA highly elevated in a similar fraction of primary tumors from the same gastrointestinal tract regions, as well as in the colon adenocarcinoma cell lines SW480 and SW1116. Further, we found M68 protein to be overexpressed in a substantial number of tumors in which gene amplification could not be detected by fluorescence in situ hybridization or quantitative genomic PCR, suggesting that overexpression of M68 may precede amplification in tumors. Finally, we find that M68 lies within a four-gene cluster that includes a novel helicase-like gene (NHL) related to RAD3/ERCC2, a plasma membrane Ras-related GTPase and a member of the stathmin family, amplification or overexpression of which may also contribute to cell growth and tumor progression.

ADP-Ribosylation Factors↗
PubMed2000

Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner.

Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.

Amino Acid Sequence↗
PubMed2000

Cytotoxicity and chromosome aberrations in vitro: experience in industry and the case for an upper limit on toxicity in the aberration assay.

The chromosome aberration assay in vitro is a useful and sensitive test for detection of genotoxins. However, aberrations can occur secondary to toxicity, with compounds that do not react with DNA and are not genotoxic in vivo. Thus, some positive results in the in vitro aberration assay are not relevant to human risk. To help evaluate the influence of toxicity, data were collected from 27 pharmaceutical and chemical companies and contract laboratories. When cytotoxicity was measured by cell counts or confluence, compounds expected to damage DNA (Category 1) generally induced aberrations without severe concomitant cytotoxicity, i.e., at cell growth 60% or more of control. The more toxic nucleoside analogues, topoisomerase inhibitors, fluoroquinolone antibiotics, antifolates, and producers of reactive oxygen were still positive with cell growth 50% or more of control. In contrast, when there was evidence that the compounds were not DNA damaging (Category 2), there was a higher proportion of toxicity-associated clastogens, with positive results at less than 50% of control cell growth. When mitotic index (MI) was used as an indicator of cytotoxicity, the pattern was less clear, although there was a tendency to more mitotic suppression with the Category 2 compounds. Overall the data indicate that a limit on toxicity, and a more accurate way of estimating it, would increase the accuracy of the assay by reducing the frequency of nonrelevant positive results with a threshold-type of dose relation. The rationale for evaluating positive results in the in vitro aberration assay, especially those associated with toxicity, is discussed, as is the need for a harmonized regulatory approach.

Animals↗
PubMed1999

Rapid method for improving slide quality in the bone marrow micronucleus assay; an adapted cellulose column procedure.

Micronuclei are routinely scored in anucleate erythrocytes in bone marrow smears stained with acridine orange. Intense fluorescence from the many nucleated cells in the preparations can interfere with micronucleus detection and cause fatigue in the reader. A method for removing nucleated cells by filtering bone marrow through cellulose packed in syringes was developed by Romagna some ten years ago, but has not been used routinely because of the excessive time needed to prepare columns. We have modified the method very simply by filling chromatography columns by pipet with a cellulose suspension. We show here that column filtration of bone marrow does not affect the numbers of micronucleated polychromatic erythrocytes (MN-PCEs) scored from mice treated with the chromosome breaking agents mitomycin C and cyclophosphamide, or the aneuploidy-inducing spindle poisons, colchicine and vinblastine. The extra preparation time is only about half an hour for a full scale micronucleus assay, and results in better slides and faster scoring.

Animals↗
PubMed1999

Occupational hazard evaluation of p-bromobenzyl bromide from tests for genotoxicity.

As part of an occupational hazard evaluation, p-bromobenzyl bromide (p-BBB) was evaluated for genotoxic activity in the Ames microbial mutagenicity assay, the alkaline elution assay for DNA strand breaks in rat hepatocytes and the in vitro chromosome aberration assay in Chinese hamster ovary cells. The compound produced equivocal results in the microbial mutagenicity assay but was negative in the alkaline elution assay for DNA strand breaks in rat hepatocytes. The compound produced weakly positive results in the in vitro chromosome aberration assay. There was substantial cytotoxicity in all three assays. It is concluded that p-BBB is weakly genotoxic.

Animals↗
PubMed1998

Fewer chromosome aberrations and earlier apoptosis induced by DNA synthesis inhibitors, a topoisomerase II inhibitor or alkylating agents in human cells with normal compared with mutant p53.

The human lymphoblastoid cell lines TK6 (normal p53) and WI-L2-NS or WTK1 (mutant p53) differ in sensitivity to killing and induction of gene mutations and chromosome aberrations by ionizing radiation. This may be related to decreased apoptosis in the cells with mutated p53, such that more damaged cells survive. We compared the response of the two cell types to various chemicals. First, to ensure that the thymidine kinase deficiency does not increase the sensitivity of TK6 tk+/- cells to mutagens, we demonstrated that they were not hypersensitive to aberration induction by altered DNA precursor pools or DNA synthesis inhibition, by aphidicolin (APC), methotrexate, hydroxyurea (HU), cytosine arabinoside and thymidine. TK6 cells were then compared with WI-L2-NS or WTK1 cells. With APC, HU, methyl methanesulfonate (MMS), ethyl nitrosourea (ENU) and etoposide (etop), TK6 cells had more apoptosis in the first two days after treatment. Fewer aberrations were seen in normal p53 TK6 cells than the mutant p53 WI-L2-NS cells, ranging from very little difference between the two cell types with MMS to very large differences with ENU and etop. For MMS and ENU we followed cultures for several days, and found that WI-L2-NS cells underwent delayed apoptosis 3 to 5 days after treatment, in parallel with published observations with ionizing radiation. WI-L2-NS cells also had a delayed increase in aberrations (up to 5 days post-treatment) when no aberrations remained in TK6 cells. Colony forming efficiency was measured for APC, MMS and ENU, and was greater in the p53 mutant cells. Our results show that normal p53 function is required for rapid and efficient apoptosis in these lymphoblastoid cells with DNA synthesis inhibitors, alkylating agents and a topoisomerase II inhibitor, and support the hypothesis that induced levels of aberrations are higher in p53 mutant cells because of a failure to remove damaged cells by apoptosis.

Alkylating Agents↗
PubMed1998

DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens.

Positive results in the in vitro assay for chromosome aberrations sometimes occur with test chemicals that apparently do not react with DNA, being negative in tests for mutation in bacteria, for DNA strand breaks, and for covalent binding to DNA. These chromosome aberrations typically occur over a narrow concentration range at toxic doses, and with mitotic inhibition. Indirect mechanisms, including oxidative damage, cytotoxicity and inhibition of DNA synthesis induced by chemical exposure, may be involved. Understanding when such mechanisms are operating is important in evaluating potential mutagenic hazards, since the effects may occur only above a certain threshold dose. Here, we used two-parameter flow cytometry to assess DNA synthesis inhibition (uptake of bromodeoxyuridine [BrdUrd]) associated with the induction of aberrations in CHO cells by DNA-reactive and non-reactive chemicals, and to follow cell cycle progression. Aphidicolin (APC), a DNA polymerase inhibitor, induces aberrations without reacting with DNA; 50 microM APC suppressed BrdUrd uptake during a 3-h treatment to <10% of control levels. Several new drug candidates induced aberrations concomitant with marked reductions in cell counts at 20 h (to 50-60% of controls) and suppression of BrdUrd uptake (<15% of control). Several non-mutagenic chemicals and a metabolic poison, which induce DNA double strand breaks and chromosome aberrations at toxic dose levels, also suppressed DNA synthesis. In contrast, the alkylating agents 4-nitroquinoline-1-oxide, mitomycin C, methylnitrosourea, ethylnitrosourea, methylmethane sulfonate and ethylmethane sulfonate, and a topoisomerase II inhibitor, etoposide, produced many aberrations at concentrations that were less toxic (cell counts >/=73% of controls) and gave little inhibition of DNA synthesis during treatment (BrdUrd uptake >/=85% of controls), although cell cycle delay was seen following the 3-h treatment. Thus, inhibition of DNA synthesis at the time of treatment is supporting evidence for an indirect mechanism of aberrations, when there is no direct DNA reactivity.

Animals↗
PubMed1998

Chromosome aberrations in vitro related to cytotoxicity of nonmutagenic chemicals and metabolic poisons.

Chromosome aberrations can occur by secondary mechanism(s) associated with cytotoxicity, induced by chemicals that do not attack DNA. Aberrations are formed from DNA double-strand breaks, and DSBs are known to be induced by nonmutagenic (Ames test negative) noncarcinogens at toxic levels [Storer et al. (1996): Mutat Res 368:59-101]. Here, 8 of 12 of these chemicals caused aberrations in CHO cells at cytotoxic doses, and often only when cell counts (survival) at 20 hr approached < or =50% of controls. Five of eight noncarcinogens (2,4,-dichlorophenol, dithiocarb, menthol, phthalic anhydride, and ethionamide) and one of two equivocal carcinogens (bisphenol A) caused aberrations, usually over a narrow dose range with steeply increasing cytotoxicity. Phthalic anhydride and ethionamide were positive only at doses with precipitate. Phenformin was negative even at toxic doses and ephedrine and phenylephrine were negative and gave little toxicity. Aberrations were also induced by metabolic poisons, 2,4-dinitrophenol, (uncouples oxidative phosphorylation), and sodium iodoacetate, (Nal; blocks ATP production). Five of the chemicals that induced aberrations in CHO cells were tested in human TK6 cells and four were positive, the fifth being equivocal. Stable aberrations (translocations) were induced in human cells by Nal. Clearly, chemicals can give "false-positive" results in the chromosome aberration assay at cytotoxic levels, though cytotoxicity does not always produce aberrations, so that further information (e.g., DNA reactivity) is needed to determine whether a result is a "false-positive." Primary DNA-damaging chemicals such as alkylators are also cytotoxic, but give strong increases in aberrations without marked initial toxicity by the measures used here, although the aberrations they induce do reduce long-term survival in colony-forming assays.

Animals↗
PubMed1997

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints.

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/beta NP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK+/- cells when tested to the limits of its solubility in the culture medium (+/- S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

Abnormalities, Drug-Induced↗
PubMed1997

Mismatch repair provokes chromosome aberrations in hamster cells treated with methylating agents or 6-thioguanine, but not with ethylating agents.

O6-Methylguanine (O6MeG) is important in induction of chromosome aberrations (abs), with the unusual property that new abs are produced in the second cycle after treatment; cells lacking repair by O6-alkylguanine DNA-alkyltransferase (AGT) have more abs at the second division (M2) than at the first (M1). These second-cycle abs are likely caused by attempted correction by mismatch repair (MMR) of O6MeG:T mispairs, since we previously showed that MMR-deficient human cells (MT1 lymphoblasts) treated with SN-1 methylating agents do not produce new abs at M2 and are resistant to killing. Here we used MMR-deficient rodent cells to examine ab induction by alkylators and by incorporated 6-thioguanine (6-tG) which produces mispairs. BrdUrd labeling was used to identify cells at first, second and third metaphase after treatment (M1, M2 and M3). MMR-deficient Chinese hamster Clone B cells were 10-fold more resistant to ab induction by methyl nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine compared to their MMR-proficient parent cells, CHO MT+. Both cell lines express AGT and can remove the methyl group from O6MeG. Clone B has twice the AGT activity of CHO MT+, but inhibition of AGT with O6-benzylguanine did not change ab induction, indicating that methylation tolerance of Clone B cells was due to defective MMR and not to increased repair of O6MeG. Confirming the importance of O6MeG in inducing abs, even when it is a minor component of the adducts induced, Clone B cells were 2-fold more resistant to ab induction by methyl methanesulfonate and dimethylsulfate, whereas they had normal sensitivity to ethyl nitrosourea and 1-ethyl-3-nitro-1-nitrosoguanidine. Clone B cells are also resistant to killing by 6-tG, and 6-tG induced few abs in MMR-deficient Clone B (6-fold lower than CHO MT+ cells). Since mispairs do not occur until the cell cycle following incorporation of 6-tG, new abs in MMR-proficient cells are expected one cell cycle later than with the methylators, i.e., at M3. As expected, in normal CHO MT+, high ab levels were seen at M3, but there was also ab induction at M2. Similarly, with methylating agents we saw higher levels of abs at M1 in the MMR-proficient CHO MT+ cells than in Clone B cells, suggesting that in the rodent cells, MMR is involved in ab formation from mispairs or modified base pairs induced in the first S-phase, such as O6MeG:C. These rodent cells thus differ from human MT1 lymphoblasts which had similar ab levels to their normal parent cells at the first metaphase after treatment with methylators.

Alkylating Agents↗