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S D Bouffler

Publications and source records attributed to S D Bouffler.

34 records · Page 2Linked to original sources
PubMed1996

The involvement of telomeric sequences in chromosomal aberrations.

Three functional elements are required for the stable transmission of eukaryotic chromosomes: replication origins, centromeres and telomeres. In the yeast Saccharomyces cerivisiae the DNA sequences defining each of these elements are known. The simplest and most widely conserved of these sequences is that of the telomere. As the name implies, the telomere is the end of a linear eukaryotic chromosome. Two of the main functions of the telomere are to prevent DNA loss as a consequence of replication and to prevent interactions with other chromosomal ends. Thus, telomeres play a major role in maintaining chromosome stability and consequently they have been considered as likely to be involved in some aspects of chromosomal aberration formation. The involvement of telomeric DNA sequences in stabilizing normal and broken chromosome ends, in "hot spots' for aberration formation and in delayed chromosomal instability will be reviewed here drawing on material presented at the Workshop and the published literature.

Animals↗
PubMed1996

Chromosomal mechanisms in murine radiation acute myeloid leukaemogenesis.

Chromosome 2 abnormalities, particularly interstitial deletions, characterize murine radiation-induced acute myeloid leukaemias (AMLs). Here, G-band analyses in CBA/H mice of early (1-6 month) post 3 Gy X-irradiation events in bone marrow cells in vivo and of karyotype evolution in one unusual AML are presented. The early event analysis showed that all irradiated animals carry chromosome 2 abnormalities, that chromosome 2 abnormalities are more frequent than expected and that interstitial deletions are more common in chromosome 2 than in the remainder of the genome. On presentation AML case N122 carried a t(2;11) terminal translocation which, with passaging, evolved into a del2(C3F3). Therefore, two pathways in leukaemogenesis might exist, one deletion-driven, the other terminal translocation-driven involving interstitial genes and terminal genes respectively of chromosome 2. As all irradiated individuals carried chromosome 2 abnormalities, the formation of these aberrations does not determine individual leukaemogenic sensitivity as only 20-25% of animals would be expected to develop AML. Similar lines of argument suggest that chromosome 2 abnormalities are necessary but not sufficient for radiation leukaemogenesis in CBA/H nor are they rate limiting in leukaemogenesis.

Acute Disease↗
PubMed1996

Chromosomal abnormalities in neutron-induced acute myeloid leukemias in CBA/H mice.

Acute myeloid leukemias (AMLs) induced in CBA/H mice by 1 MeV fission neutrons have been examined for chromosomal abnormalities by G-band analysis. In common with X-ray- and alpha-particle-induced AMLs in CBA/H mice, more than 90% (16/17) of the myeloid leukemias had chromosome 2 abnormalities, in this case, all interstitial deletions. Chromosome 2 breakpoints were not wholly consistent, but clustering in three specific G-band regions was observed. Very distal (H-region) breakpoints were more common in the neutron AMLs than in X-ray- or alpha-particle-induced leukemias. These data indicate that neutron-induced AMLs in CBA/H mice are not characterized by a specific chromosome deletion but that a variety of chromosome 2 deletion types are associated with the disease. Trisomy of chromosome 1(12.5% AMLs) and aneusomy of chromosomes 6 (31% AMLs) and Y (37.5% AMLs) were noted. While chromatid breakage was observed occasionally in neutron-induced AML, no clear indications of persistent chromosomal instability or high levels of stable chromosomal change were apparent.

Acute Disease↗
PubMed1995

Spontaneous and ionizing radiation-induced chromosomal abnormalities in p53-deficient mice.

Chromosomal abnormalities have been assessed in p53-deficient mice. The in vivo frequency of spontaneous stable aberrations in bone marrow cells was elevated by approximately 20-fold in p53 nulls and 13-fold in p53 heterozygotes compared to wild-type. No excessive induction of stable aberrations by gamma-irradiation was observed, but p53 deficiency resulted in excess radiation-induced hyperploidy (> 10-fold wild-type frequency). No influence of p53 genotype on sister chromatid exchange or G2 chromatid damage was observed in mitogen-stimulated spleen cell cultures; however, a p53 effect on postirradiation mitotic entry was seen. Abnormalities in chromosome segregation and mitotic delay following irradiation in p53-deficient mice suggest a G2-M checkpoint role for p53 and are broadly consistent with data on tumorigenesis in these animals.

Aneuploidy↗
PubMed1992

Characterisation and correction of a mammalian cell mutant defective in late step of base excision repair.

An Indian muntjac cell line, SVM, is unusually sensitive to cell killing induced by a range of alkylating agents. Cells transfected with the Escherichia coli ada gene or human genomic DNA have allowed the response of SVM to alkylating agents to be dissociated into two distinct components. Thus, in SVM, which expresses very low levels of alkyltransferase (AT), O6-alkylguanine appears to be the major cytotoxic, clastogenic, and recombinogenic lesion following exposure to agents such as methylnitrosourea (MNU). However, SVM is also very sensitive to agents such as dimethylsulfate (DMS), which produce only very low levels of O6-methylguanine damage. Sensitivity to DMS resides in an inability to complete base excision repair, with the appearance of persistent single-strand DNA breaks (SSBs), and does not appear to involve defects in glycosylase, apurinic/apyrimidinic endonuclease, or DNA ligase activities. Another, possibly related, phenotypic trait in SVM is its limited ability to ligate transfected linear plasmid DNA. Transfectants of SVM, harboring human DNA sequences, show a significant correction of DMS-induced cytotoxicity and clastogenicity and a reduction in the levels of DMS-induced DNA SSBs. The DMS-resistant transfectants have an increased ability to ligate linear plasmid DNA, and also express AT, making these lines resistant to alkylating agents such as MNU. These results suggest that cells possess a mechanism that regulates AT expression, plasmid break-joining ability, and certain aspects of base excision repair. Transfectants of SVM containing human DNA provide a means to isolate genes involved in a coordinate response to alkylation damage.

Alkylating Agents↗
PubMed1992

Cockayne's syndrome fibroblasts are characterized by hypersensitivity to deoxyguanosine and abnormal DNA precursor pool metabolism in response to deoxyguanosine or ultraviolet light.

New cellular traits of Cockayne's syndrome (CS) associated with DNA precursor metabolism have been identified, namely, hypersensitivity to the toxicity of low concentrations of deoxyguanosine (dG) and abnormal changes in deoxyribonucleotide (dNTP) pools in response to dG or UV. dG treatment results in similar ribonucleotide pool changes in wild-type and CS cells, i.e., GTP levels increase at least twofold. However, the changes in the pool size of the purine deoxyribonucleotides are significantly different; in wild-type cells dATP and dGTP pools increase threefold, but remain unchanged in CS. The mechanism by which dG kills CS cells is not clear, but unlike the inherited purine nucleoside phosphorylase deficiency disease, the toxicity of dG is not due to the accumulation of dGTP and the consequent feedback inhibition of ribonucleotide reductase. UV induces different dNTP pool changes in CS and wild-type cells. In wild-type cells dTTP, dCTP, and dATP pools increase three- to fivefold within 4 h of irradiation, while the dGTP pool contracts. In CS cells, only the dGTP pool expands (four- to sixfold), while the other three contract. Each of these new phenotypic traits, together with UV sensitivity, is coordinately corrected in the complementing proliferating CSA x CSB hybrid cells.

Cell Division↗
PubMed1992

Mammalian cells share a common pathway for the relief of DNA replication arrest by O6-alkyl guanine, incorporated 6-thioguanine and UV photoproducts.

We previously reported the cloning of a mammalian gene that restores UV resistance to a postreplication recovery defective and mex- Indian muntjac mutant cell line, SVM, by improving daughter-strand DNA replication on a UV-damaged template. The improved replication was, however, found to be error-prone, as judged by a hypermutable phenotype (Bouffler et al. (1990) Somatic Cell Mol. Genet., 16, 507-516). We now report that this gene also increases the resistance of SVM to the cytotoxic effects of methyl- and ethyl-nitrosourea, though not to dimethyl sulphate, by a similar postreplication recovery process. The gene does not increase the activity of O6-alkylguanine-DNA-alkyltransferase in the cell. We conclude that at least one mechanism of postreplication recovery in mammalian cells allows UV photoproducts and O6-alkylguanine lesions to be tolerated by the replication complex. The fact that the gene also confers resistance to 6-thioguanine suggests that, once incorporated, this base analogue can disrupt normal DNA replication and that a single mechanism can allow replication to proceed beyond 3 diverse DNA lesions.

Alkylating Agents↗
PubMed1991

Abnormal sister-chromatid exchange induction by 3-aminobenzamide in an SV40-transformed Indian muntjac cell line: relationships with DNA maturation and DNA-strand breakage.

In SVM cells, an SV40-transformed line of Indian muntjac fibroblasts, levels of sister-chromatid exchanges are known to be abnormally high after UV-irradiation or alkylation. The SVM line is also known to have a defect in the processing of DNA-strand breaks. Sister-chromatid exchange in other cells is known to be stimulated by the poly(ADP-ribose) transferase inhibitor, 3-aminobenzamide, which also retards DNA-break sealing. Sister-chromatid exchanges in SVM cells are found to be hypersensitive to 3-aminobenzamide, or to nicotinamide deprivation which similarly inhibits poly(ADP-ribosyl)ation; DNA-strand breaks are likewise induced by 3-aminobenzamide. Bromodeoxyuridine, needed to detect sister-chromatid exchanges, is more toxic to SVM cells and itself induces sister-chromatid exchanges to a greater extent than it does in normal muntjac cells. However, in contrast to the situation reported for other cell types prone to sister-chromatid exchange (the Chinese hamster ovary mutant EM9 and human Bloom's Syndrome cells), SVM cells do not show an abnormal delay in DNA maturation when, under the influence of bromodeoxyuridine and 3-aminobenzamide, they show a high level of sister-chromatid exchange. The mechanism by which BrdU exerts its effects can largely be explained in terms of familiar effects on deoxyribonucleotide pools and DNA integrity. 3-Aminobenzamide, however, induces sister-chromatid exchanges in SVM cells by another mechanism.

Animals↗
PubMed1990

Molecular cloning of a mammalian gene involved in the fixation of UV-induced mutations.

A mammalian DNA damage tolerance gene has been isolated by DNA transfection and cosmid rescue. Following cotransfection of mouse genomic DNA and pSV2neo into SVM, the UV hypersensitive mutant Indian muntjac cell line, clones with a 1.7 to 2.0-fold greater D37 value for UV killing were isolated. This trait was carried through three rounds of transfection. A neo gene and flanking sequences from a tertiary transfectant were cloned by cosmid rescue. The cosmid clone confers UV resistance to SVM and improves the ability of the cell to replicate UV damaged DNA. This replication appears to be error-prone; UV-induced 6-thioguanine-resistant mutants occur four to fivefold more frequently than in SVM or a wild-type Indian muntjac line. Thus, the gene isolated is not homologous to that defective in SVM. We believe that this is the first mammalian gene to be isolated that is directly involved in mutation fixation.

Animals↗
PubMed1990

Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line.

DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.

Animals↗
PubMed1990

Changes in muntjac fibroblasts associated with the acquisition of cadmium resistance. A pre-resistance, transitional and post-resistance study.

A series of cell lines with different levels of resistance to continuous cadmium exposure has been developed from an immortal but non-transformed muntjac fibroblast cell line. Concentrations accepted in their culture medium range from 0.1 microM for the cadmium sensitive parent line to 5 microM for the intermediate "cadmium-tolerant" line, to 5, 10, 20 and 50 microM for the four "cadmium-resistant" lines. The present paper follows the morphological changes which accompanied the development of resistance through a 20-month pre-resistance period, a relatively abrupt 6-week transitional period and a 3-year post-resistance period, during which time levels of cadmium resistance were increased. Initial changes which led to the cadmium-tolerant CR5 cell line included (i) increased efficiency in autophagocytosing damaged cell components and in ridding the cell of residual waste materials, (ii) a reduction in fluid filled vacuoles and (iii) improved recycling and/or replacement of cadmium-damaged cell membrane. With the advent of cadmium resistance the intracellular damage necessitating these activities disappeared, yet the series of changes which occurred included a massive build-up of Golgi and the appearance of a trans-Golgi tubular network in addition to cytoskeletal and membrane changes. Though metallothionein levels are greater in the cadmium-resistant variants, their increase appears inadequate on their own to account for the high levels of resistance. The post-resistance changes which accompanied each step-up in cadmium resistance included further membrane and glycocalyx changes, in addition to continued increases in Golgi bodies and tubular network. This paper details the morphological changes which occurred throughout the 5-year period, tests the direct dependence of each on the presence of cadmium and examines their possible contribution to a cadmium protective mechanism.

Animals↗
PubMed1989

Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines.

The responses of two Indian muntjac cell lines to two monofunctional alkylating agents were investigated. An SV40-transformed line (SVM) had an increased sensitivity to cell killing when compared to the other, euploid line (DM) after exposure both to methyl nitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibited higher frequencies of sister chromatid exchanges (SCEs) following alkylation. The hypersensitivity of SVM to DMS correlates with the defective repair of single-strand breaks that results in the generation of long-lived breaks in the DNA following exposure, leading eventually to the formation of chromosome aberrations. In contrast no difference is seen in the formation of long-lived breaks in the DNA of SVM and DM after treatment with biologically relevant doses of MNU; in this case hypersensitivity may be due to the loss of O6-alkylguanine-DNA-alkyltransferase activity. The conclusion that the hypersensitivites of SVM to MNU and to DMS have different molecular bases is supported by transfection of SVM with plasmids containing the protein coding region of the Escherichia coli ada+ gene; subsequent expression within the cell corrects its hypersensitivity to the cytotoxic and SCE-inducing effects of MNU but has very little influence upon the lethality, SCE induction or the repair of long-lived DNA strand breaks after exposure to DMS.

Alkylating Agents↗
PubMed1989

CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers.

The CHO mutant UV61 was previously assigned to complementation group 6 of UV-sensitive rodent cell mutants. UV61 is less sensitive to killing by UV radiation than mutants such as UV5, which is highly defective in the incision process that acts on UV-induced lesions. The D37 for cell survival is approximately 4 J/m2 for UV61, compared with 10 J/m2 for the parental AA8 line and approximately 2 J/m2 for UV5. Similarly, mutation induction at the hprt and aprt loci shows an intermediate response to UV61. In a post-replication recovery assay, the kinetics of maturation of pulse-labelled nascent DNA were normal after UV irradiation in UV61. Data from alkaline elution and alkaline unwinding assays showed that the rates of break accumulation and resealing, measured 0-120 min after irradiation, were also normal in the mutant. This repair incision correlated with the rapid, normal removal of pyrimidine(6-4)pyrimidone photoproducts in UV61 measured using a radioimmunoassay that is specific for this class of damage. In contrast, after exposure to 10 or 15 J/m2, no detectable removal of cyclobutane dimers from DNA was found in UV61 while AA8 cells removed 32% by 24 h. We suggest that the mutation in UV61 specifically lowers the affinity of a repair protein for cyclobutane dimers, which are also inefficiently removed from the bulk DNA of normal CHO cells. The resistance of UV61 to killing by the direct acting chemical 7-bromomethylbenz[a]anthracene was only slightly greater than that of UV5, indicating defective repair of bulky chemical adducts in addition to cyclobutane dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Cadmium induced changes in cell organelles: an ultrastructural study using cadmium sensitive and resistant muntjac fibroblast cell lines.

A detailed electron microscopy study of cadmium sensitive and resistant muntjac fibroblast cell lines has identified a wide range of intracellular damage following exposure to cadmium. Damaged organelles included cell membrane, mitochondria, Golgi cisternae and tubular network, chromatin, nucleoli, microfilaments and ribosomes. Although cell membrane damage was generally the earliest indication of adverse cadmium action, particularly with continuous cadmium exposures, cells could tolerate extensive membrane loss. Mitochondrial distortion and some damage to Golgi was also tolerated. The turning point at which cadmium became lethal was generally marked by a cascade of events which included damage to both nuclear and cytoplasmic components. These results for fibroblasts are discussed and compared with damage reported in other types of cells.

Animals↗
PubMed1988

Parallel development of cadmium resistance and in vitro transformation in cultured Indian muntjac cells.

The development of cadmium resistance in an Indian muntjac cell line has been investigated. The parent cell line is highly sensitive to cadmium ions. Resistance was obtained by continuous growth of cells in low levels of cadmium with stepwise increments. Four cell lines were developed with resistances of between 50- and 200-fold greater than that of the parental line. Early in the development of resistance an unstable cell line displaying extensive chromosomal rearrangement and an elevated sister chromatid exchange frequency was identified. The more stable resistant lines produced from this original cell line have normal karyotypes. Having passed through the initial period of genome rearrangement the resultant cells acquired several characteristics of morphologically transformed cells. It is concluded that long-term exposure to low levels of cadmium can transform cells in vitro concurrently with their acquiring cadmium resistance.

Animals↗