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W G Thilly

Publications and source records attributed to W G Thilly.

At least 91 records · Page 5Linked to original sources
PubMed1989

Direct measurement of mutational spectra in humans.

By combining high fidelity in vitro DNA amplification and mutant DNA sequence separation by denaturing gradient gel electrophoresis, we are able to directly observe mutational hotspots in human genomic DNA. Our technological development has progressed through the stage of identifying mutant sequences in independently derived, 6-thioguanine-resistant human B cells. We are now analyzing uncloned, complex populations derived from several thousand 6-thioguanine-resistant cells and report preliminary data concerning the mutational spectra of benzo[a]pyrene diol epoxide and ultraviolet light in exon 3 of the hypoxanthine-guanine phosphoribosyltransferase gene. In addition, the approach appears to be general for any gene sequence for which a means to select mutants exists. The more global need to eliminate phenotypic selection is, however, our primary impetus. Our analysis leads us to conclude that no known in vitro DNA polymerase has sufficient fidelity to permit direct observation of unselected mutants. Therefore, an additional change in technology will be necessary to observe nonselected mutant DNA sequences at the low frequencies found in human tissues.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

Growth of mammalian cells at high oxygen concentrations.

Oxygen, although essential to the growth of mammalian cells in vitro and in vivo, has been widely reported to be toxic at concentrations at or above the oxygen concentration in culture medium equilibrated with air (approximately 200 microM). We were therefore surprised to note that a diploid human B-cell line (TK6) was able to proliferate normally while exposed to 380 microM-oxygen. This observation was extended to Vero (African Green monkey kidney) cells, and Sp2/0-derived murine transfectomas producing antibody. Using an experimental system with a high capacity for oxygen transfer, we determined the growth rates of the three cell lines at controlled oxygen concentrations ranging from 80 microM to 910 microM. Each of these cell types was able to grow normally at oxygen concentrations up to 360-380 microM. At oxygen concentrations above 380 microM, a significant increase in the apparent doubling times of the cells was observed. No adverse effect of oxygen on TK6 cell survival was seen for concentrations ranging from 60 microM to 410 microM. We conclude that exponential growth at nearly maximum growth rates was observed for Sp2/0-derived cells, TK6, and Vero cells at constant oxygen concentrations up to 400 microM (twice air saturation). These findings have substantial implications for process control in the production of cells and cell-derived materials. The intrinsic oxygen transfer rate of any cell culture vessel can be increased severalfold by raising the oxygen gas concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Towards development of techniques to observe mutational spectra in human tissue samples.

We have devised a protocol with the resolving power to uncover mutational spectra within DNA sequences from human genomic DNA. The protocol is a combination of: (a) in vitro DNA amplification of the desired DNA sequence; (b) denaturing gradient gel electrophoresis of the amplified DNA to separate the wild type and mutant sequences; (c) isolation of the mutant sequence(s); (d) determination of the nature of the base-pair change(s). Data based on reconstruction experiments using exon 3 of the human hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene suggest that the protocol can detect mutant sequences present at mutant fractions equal to or greater than 10(-4).

Base Sequence↗
PubMed1988

Mapping large spontaneous deletion endpoints in the human HPRT gene.

In an attempt to understand the nature, frequency, and molecular origin of spontaneous mutations in human cells, we have analyzed 85 independent, spontaneous HPRT- human B-lymphoblast clones with particular emphasis on the determination and characterization of large structural alterations (i.e., deletions, insertions, duplications, etc.). Southern blot analysis using a full-length HPRT cDNA probe revealed that 39% (33/85) of these spontaneous mutants contained alterations affecting different regions of the gene. 12% (10/85) were total gene deletions, 25% (21/85) involved alterations with one or both endpoints intragenic to HPRT, and 2% (2/85) showed wild-type banding patterns with an additional hybridizing band. To further address the positional behavior of these alterations, the endpoints of the large deletions were mapped to specific exon/intron regions by hybridization of Southern blots with a series of HPRT exon-specific probes. This analysis revealed a disproportionate number of endpoints within the 3' portion of the gene. These findings are discussed in relation to the positional specificity of large alterations in human cells and the use of such an analysis for assessing the molecular mechanism(s) responsible for their production.

B-Lymphocytes↗
PubMed1988

Binding of cisplatin to specific sequences of human DNA in vitro.

Cisplatin was reacted with a 184-base-pair sequence, exon 3, of human HPRT DNA in vitro. The binding sites were mapped by a primer extension method with T4 DNA polymerase and radioactive dCTP. Binding sites of cisplatin were indicated by the lengths of synthesized polynucleotides as determined by gel electrophoresis. Neighboring GG dinucleotides were highly preferred sites of binding by cisplatin, while less binding was noted to GXG, GA, AAA, and GXA. Analysis by densitometry revealed a 5-fold difference in binding among the GG sequences. The relative binding to a GGG sequence exceeded that of a GGGGGG sequence, suggesting that the number of Gs in a run did not determine the relative binding.

Base Sequence↗
PubMed1988

Looking ahead: algebraic thinking about genetics, cell kinetics and cancer.

The cancer cell phenotype is modelled as a set of necessary genetic changes containing from one to three changes for any particular set of developmentally identical cells (tissue). This analysis suggests that a person in whose tissue an early genetic change occurs during ontogeny will perforce have a markedly higher number of cells in single sectors containing the necessary set of genetic changes. It is hypothesized that this numerical expectation is a dominant factor in determining the probability that a particular person will develop a tumour in a particular tissue.

Cell Division↗
PubMed1988

Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich.

The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning. DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation. The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing. Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning. HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397. We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene and contains a low-temperature melting domain of approximately 100 bp. HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3. We report the separation of HPRTMunich from the wild-type sequence using DGGE. In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule. The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HPRT cDNA was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1987

Killing and mutation of human lymphoblast cells by aflatoxin B1: evidence for an inducible repair response.

Diploid human lymphoblast cells exhibit apparent saturation of mutation induced by exposure to aflatoxin B1, despite a linear increase in the amount and proportion of the aflatoxin-DNA adducts formed. The saturation is neither a cell cycle phenomenon nor a result of a genetically heterozygous population. Examination of the biphasic nature of aflatoxin-DNA adduct loss in vivo shows initial, rapid removal of all adduct species, followed by a slow loss of the aflatoxin-N7-guanine adduct alone. We hypothesize that these data reveal two modes of adduct loss in these cells. The first is an inducible, error-free system that is short-lived, turning off as adduct levels fall below the induction threshold of some 1000 total adducts/cell. The second loss is slower and results from spontaneous depurination of remaining aflatoxin-N7-guanines. Our data are in agreement with the possibility that apurinic sites thus generated are responsible for the mutation observed. A major paradox arises from the fact that aflatoxin-related premutagenic depurinations are estimated to be only 10% of the number of spontaneous depurinations estimated by others to occur in human cells in a 1-h period.

Aflatoxin B1↗
PubMed1987

Internal standards for survival: increasing the accuracy for human cell mutation assays.

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation or transformation assays and is a necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.

4-Nitroquinoline-1-oxide↗
PubMed1987

Commercial hickory-smoke flavouring is a human lymphoblast mutagen but does not induce lung adenomas in newborn mice.

Commercial aqueous wood-smoke flavouring induced significant increases in the 6-thioguanine resistance mutation frequency of TK6 human lymphoblasts at 0.1 microliter flavouring/ml of cell suspension. This corresponds to 6 micrograms/ml of dissolved 'solids' as determined by fully drying the aqueous flavouring in a vacuum desiccator. In AHH-1 human lymphoblasts, which contain a cytochrome P-450 monooxygenase system, mutations were induced at 0.3 microliter/ml, corresponding to 18 microliters/ml of dissolved 'solids'. The flavouring did not induce 8-azaguanine resistant mutations in Salmonella typhimurium at concentrations up to 1.5 microliter/ml. At higher concentrations the flavouring was toxic to bacteria. The flavouring did not induce lung adenomas or other tumours in newborn mice when injected ip with total doses of up to 26 microliters over a 3-wk period. Toxicity to the kidney, colon and rectum was observed in some mice at 15 wk of age.

Adenoma↗
PubMed1987

Chemical and toxicological characterization of residential oil burner emissions: II. Mutagenic, tumorigenic, and potential teratogenic activity.

Extracts of effluents from a modern residential oil burner have been evaluated in several toxicological assay systems. Bacterial mutagens were detected in extracts from both the particulate and vapor phase emissions. Effluents from continuous operation were an order of magnitude less mutagenic than those from cyclic (5 min on, 10 min off) operations. No difference in the yield of bacterial mutagens per gram of fuel burned was found between cyclic operation under low and moderate sooting conditions. On the basis of elution behavior from alumina it appeared that the bacterial mutagens collected from high sooting effluents were more polar than those from low sooting effluent. An extract that was mutagenic in bacteria did not induce a significant increase in mutation frequency to human lymphoblasts. No evidence of tumorigenicity was observed in a limited number of newborn mice after IP injection of effluent extract when compared to historical control data. Putative nonmutagenic teratogens were detected in effluent using an attachment inhibition assay. The level of these agents was reduced in effluents from continuous oil burner operation.

Air Pollutants↗
PubMed1986

Microsomal activation of fluoranthene to mutagenic metabolites.

The in vitro metabolism of fluoranthene (FA) was assessed by incubating 3-[3H]FA, the synthesis of which is described, with rat hepatic microsomal enzymes. Several metabolites including the FA 2,3-diol, FA 2-3,-quinone, 3-OH-FA, 1-OH-FA, and 8-OH-FA were isolated by high-pressure liquid chromatography and identified by comparison of chromatographic properties and uv-visible spectra with those of synthetic standards. The major metabolite produced over the FA concentration range studied (23-233 microM) was FA 2,3-diol, accounting for 29-43% of the total extractable metabolites. This diol was characterized further by high-resolution mass spectroscopy and H-NMR and determined to be identical in structure to the trans-2,3-dihydroxy-2,3-dihydrofluoranthene. The FA 2,3-diol, syn and anti 2,3-diol-1,10b-epoxides, FA 2,3-quinone, and FA 7,8-diol were all shown to be mutagenic toward Salmonella typhimurium TM677. The FA 1,10b-diol and syn and anti FA 1,10b-diol-2,3-epoxides were not mutagenic. The epoxide hydrolase inhibitor, 3,3,3-trichloropropylene oxide, markedly reduced the mutagenic potency of FA while concurrently inhibiting FA 2,3-diol production but not overall FA metabolism. These results suggests that a major metabolic activation pathway of FA resulting in the production of mutagenic species involves the formation of the FA 2,3-diol and the subsequent oxidation of this diol to a FA 2,3-diol-1,10b-epoxide. Another minor activation pathway with mutagenic endpoints may involve the formation of the 7,8-diol.

Animals↗
PubMed1986

Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine.

Isogenic variants resistant to alkylating agents have been isolated from the human lymphoblast cell line TK6. The cell lines may be divided into four classes on the basis of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The sensitive TK6 parental line shows a 37% survival after 45-min exposure to 0.04 microM MNNG; the three classes of more resistant mutants show 37% survival after 45-min exposure to 2 microM (MF lines), 6 microM (MT lines), and greater than or equal to 10 microM (MX line) MNNG. A representative MF line, MF1, is resistant to both killing and mutation by MNNG or N-methyl-N-nitrosourea. An MT clone, MT1, is highly resistant to killing but hypermutable by MNNG. The MT1 line, like the parental TK6, does not remove O6-methylguanine adducts from the DNA. Our data are consistent with the hypothesis that the MT1 line possesses a nonexcision pathway of defense against killing by alkylating agents. Rather than preventing alkylation of DNA or removing alkylated adducts, the MT1 cells appear to be tolerant of the adducts that are not removed from the DNA.

Alkylation↗
PubMed1986

Use of gradient denaturing gels to determine mutational spectrum in human cells.

Based on the fact that mutagens induce specific patterns of gene mutations, this paper outlines a method to allow discrimination among mutagen-treated populations. The technique should allow direct screening of human tissue for genetic change, using human peripheral blood lymphocytes deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase. The method is based on gradient denaturing gel electrophoresis, which separates short DNA molecules according to their melting properties. The melting behavior of DNA fragments is extremely sequence-dependent, and DNAs with single basepair substitutions often migrate differently. Even DNA fragments with the same basepair substitutions at different locations in the molecule have been resolved. Gradient-denaturing gel electrophoresis has the capacity to separate mutant DNA on the basis of the nature and position of the mutation.

Base Sequence↗
PubMed1986

Spontaneous and induced chromosomal aberrations and gene mutations in human lymphoblasts: mitomycin C, methylnitrosourea, and ethylnitrosourea.

The concentration-dependent mutagenic, clastogenic, and cytocidal activities of mitomycin C (MC), methylnitrosourea (MNU), and ethylnitrosourea (ENU) were measured in the human lymphoblast cell line TK6. For treatments resulting in fewer than 2 lethal hits, MNU, ENU, and MC gave rise to apparently linear dose-response curves for gene mutations (hgprt and tk genes) as well as for chromosomal aberrations. The numbers of induced mutants at the tk and hgprt loci were similar between the two loci for each compound. However, the ratio of mutagenic activity relative to the clastogenic activity (aberrations/cell) was lowest for mitomycin C, intermediate for methylnitrosourea, and highest for ethylnitrosourea. These results confirm in human cells the general observation that the processes of mutagenesis and clastogenesis are nonidentical: compounds vary independently in their mutagenic and clastogenic potentials.

Cell Line↗
PubMed1986

Studies of mutagenicity and clastogenicity of 5-azacytidine in human lymphoblasts and Salmonella typhimurium.

5-Azacytidine (5-AzaC) induced mutation in the TK+/- human lymphoblastoid line, TK6, at both the thymidine kinase (tk) locus as measured by resistance to trifluorothymidine (F3TdR), and the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, as measured by resistance to 6-thioguanine (6TG). F3TdRR and 6TGR mutant fractions induced by 5-AzaC were observed after a normal phenotypic expression time and remained stable. Interestingly, 5-AzaC was 5-10 times more mutagenic at the tk locus than the hgprt locus. However, F3TdRR colonies from 5-AzaC-treated cultures behaved like TK-deficient mutants induced by other chemical mutagens. The TK or HGPRT phenotype had no effect on the toxicity of 5-AzaC, thus eliminating differential toxicity as a potential cause for the observed higher mutability at the tk locus. 5-AzaC did not induce F3TdRR cells in the parental TK+/+ lymphoblastoid line, indicating that 5-AzaC-induced F3TdRR variants were not due to a dominant alteration in gene expression. 5-AzaC did not induce chromosomal aberrations in TK6 cells, eliminating clastogenic events as a potential cause for the higher mutability at the tk locus. 5-AzaC was also found to be mutagenic in a forward mutation assay to 8-azaguanine resistance in Salmonella typhimurium.

Azacitidine↗