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Biomedical subjects

W G Thilly

Publications and source records attributed to W G Thilly.

158 records · Page 9Linked to original sources
PubMed1977

Cell density dependence of focus formation in the C3H/10T1/2 transformation assay.

Some of the dynamics of neoplastic transformation in vitro have been studied with the use of benzo(a)pyrene as the carcinogen in the C3H/10T1/2 morphological transformation assay. Experiments that involved the dsipersion of cells into new culture dishes at various times after carcinogen treatment have shown that no change in the fraction of potentially transformed cells occurs while cultures grow to form a confluent monolayer, that little or no change in the fraction of potentially transformed cells occurs for approximately 3 weeks after confluence is attained, and that this fraction increases rapidly some 7 weeks after BP treatment. When confluent benzo(a)pyrene-treated cultures are dispersed in new culture dishes prior to the onset of growth toward focus formation, the formation of transformed foci is suppresssed at high cell densities of seeding. This phenomenon is independent of the total number of divisions undergone by cells after treatment. We suggest that phenotypic expression of morphological transformation is dependent on colony interactions in the C3H/10T1/2 system, which we do not yet understand, but which are independent of time posttreatment either in cell generations or absolute time.

Animals↗
PubMed1976

Concentration-dependent mutation of diploid human lymphoblasts by methylnitronitrosoguanidine: the importance of phenotypic lag.

The mutation of diploid human lymphoblasts by methylnitronitrosoguanidine (MNNG) was measured over the range of 0--45 ng of MNNG/ml of of medium. We found a 12-day lag in the phenotypic expression of 6-thioguanine resistance; the occurrence of this lag was independent of MNNG concentration. We hypothesize that the unexpectedly long lag period reflects a requirement for the loss of previously existing molecules of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) after mutation at the HGPRT locus.

Cell Survival↗
PubMed1976

Mutation of human lymphoblasts by methylnitrosourea.

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8-12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (less than 0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 mug 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.

Cell Line↗
PubMedDate not supplied

Variance estimation in single-cell mutation assays: comparison to experimental observations in human lymphoblasts at 4 gene loci.

The performance of mutation assays with single cells involves a series of separate steps beginning with the induction of mutant cells and ending with the counting of mutant and wild-type colonies. The variation among identically treated cultures is here modeled as arising from 3 sources: (1) the number of mutant cells surviving treatment, (2) the number of mutant cells sampled in steps of sampling and growth required in assays involving phenotypic lag, and (3) the number of mutant and nonmutant colonies actually observed. The arithmetical statements describing the expectation of variance from each step are presented and used to provide means to calculate an expected overall variance for typical experiments. The model is then tested by comparing its predictions with the observed mutant fractions in human lymphoblastoid cells at the loci coding for 6-thioguanine, ouabain, podophyllotoxin, and 5,6-dichlororibofuranosyl benzimidazole resistances. The model is found to have excellent predictive qualities and should be useful in experimental design of studies involving induction of rare variants in single-cell systems.

Cells, Cultured↗
PubMedDate not supplied

Cell-cycle dependent mutation of human lymphoblasts: bromodeoxyuridine and butyl methanesulfonate.

Cells of the human lymphoblast line WI-L2 and its derivative TK-6 were synchronized by centrifugal elutriation and cell-cycle dependent mutation to 6TGR (HPRT) and OUAR (Na+, K+ ATPase) measured. Bromodeoxyuridine induced 6TGR and OUAR mutations within S phase while butylmethyl-sulfonate induced mutation displayed no cell-cycle dependence. The data indicate that centrifugal elutriation is a facile means to obtain a useful degree of synchrony for these cell lines.

Bromodeoxyuridine↗
PubMedDate not supplied

The aza-arenes as mutagens for Salmonella typhimurium.

Several unsubstituted aza-arenes have been found to be more mutagenic to Salmonella typhimurium than their corresponding parent hydrocarbons. In most cases, the activity of these compounds depended on the presence of a post-mitochondrial supernatant for metabolic activation, although acridine was mutagenic only in the absence of such an activating system. An examination of the effect of the metabolizing system's concentration on mutagenicity showed that quinoline, benzo[f]quinoline, and phenanthridine have different optima. In an attempt to uncover active intermediates in aza-arene metabolism, N-oxides of quinoline and phenanthridine were synthesized and found to be non-mutagenic, and coincubation with the epoxide hydrase inhibitor trichloropropylene oxide did not affect the mutagenic activity of quinoline or phenanthridine.

Animals↗
PubMedDate not supplied

Mutational spectrum of chromium(VI) in human cells.

The mutational spectrum of K2Cr2O7 on the low melting domain within exon 3 of hypoxanthine guanine phosphoribosyl transferase (hprt) gene was obtained from 6-thioguanine-resistant (6TGr) human lymphoblasts (TK6). Using a combination of high-fidelity polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE), we found four chromium(VI) induced hotspots in a 104-bp target sequence. The hotspots were C:G-->A:T at bp 243, representing 4.5% of 6TGr mutants; A:T-->T:A at bp 247 (2.0%); G:C-->A:T at bp 289 (2.5%) and C:G-->T:A at bp 312 (4.0%). Mutational hotspots at bp 243 have previously been determined for hydrogen peroxide (C:G-->G:C) and benzo[a]pyrene diol epoxide (C:G-->A:T). Two of the hotspots, bp 243 and 289, arose in a common quadruplet 5'-ACAT-3' in the untranscribed and transcribed strands respectively.

Base Sequence↗
PubMedDate not supplied

DNA amplification in vitro using T4 DNA polymerase.

We have evaluated in vitro DNA amplification by polymerase chain reaction using either T4 DNA polymerase or Klenow fragment of Escherichia coli DNA polymerase I. Both polymerases under optimal salt conditions permit efficient amplification of exon 3 of the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene from human genomic DNA and from plasmid containing the HPRT cDNA. DNA sequences amplified from human genomic DNA, using two 20-nucleotide primers flanking the ends of the exon, showed a marked difference between the two polymerases. T4 DNA polymerase yielded only the expected amplified DNA fragment, whereas Klenow fragment produced many lower-molecular-weight bands in addition to the expected DNA fragment. On the basis of the reported fidelity of in vitro DNA synthesis using Klenow fragment and T4 DNA polymerase, it is expected that the latter will create substantially fewer errors during the amplification process. For these reasons, T4 DNA polymerase should be particularly valuable for amplification of sequences present at a very low frequency requiring many cycles of amplification to be detected.

DNA↗
PubMedDate not supplied

Mutation assay in diploid human lymphoblasts: methodological aspects.

The protocol for a recently developed quantitative assay for mutation at the hgprt locus of human lymphoblasts is presented. Practical problems affecting ease of performance and reliability are discussed with the aim of making the assay available for assessment and possible use in other laboratories.

Cell Line↗