Biomedical subjects

M Applegate

Publications and source records attributed to M Applegate.

10 recordsLinked to original sources

PubMed1992

In vitro mammalian mutagenesis as a model for genetic lesions in human cancer.

Recently in vitro assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Questions have also been raised concerning the relevance of rodent assays to human risk. In vitro assays using mammalian cells can detect most types of genetic lesions thought to be important in human malignant disease. Molecular and cytogenetic analyses of mutations induced by a variety of genotoxic compounds at the heterozygous thymidine kinase locus in mouse lymphoma cells indicate that this in vitro assay does indeed register the range of genetic lesions recently found in a wide variety of human tumors. The types and complexity of the induced lesions are reflected in mutant colony phenotype in a compound-specific fashion. These studies point to the use of appropriate in vitro mammalian mutagenesis assays as new model systems for dissecting the genetic lesions important in human carcinogenesis, and as a means of determining the potential for compounds to induce such lesions.

Animals↗

PubMed1992

Comparison of the effects of Wyeth-14,643 in Crl:CD BR and Fisher-344 rats.

Wyeth-14,643 (WY) belongs to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation and hepatocellular carcinoma in rodents. Based on a review of bioassay data, a relationship appears to exist between peroxisome proliferating compounds and Leydig cell adenoma formation. Most rat bioassays with peroxisome proliferators have been conducted in the Fisher-344 (F344) rat, which has a high spontaneous incidence of Leydig cell adenomas. Thus, it was necessary to use an alternative animal model to investigate this relationship further. Therefore the Crl:CD BR (CD) rat, which has a low spontaneous Leydig cell adenoma incidence, was chosen for a 2-year feeding study with WY. Before initiating this 2-year feeding study, it was necessary to investigate whether any strain differences existed between CD and F344 rats with respect to WY-induced peroxisome and cell proliferation. In this study, male CD and F344 rats were fed diets containing 0, 50, or 1000 ppm WY for 21 days. Peroxisome proliferation in the liver and testis was determined biochemically by measuring beta-oxidation activity and was confirmed ultrastructurally. Serum hormone levels and cell proliferation rates in the liver and testis were also measured. In addition, basal beta-oxidation activity and cell proliferation rates were compared between the CD and F344 rats. A significant decrease in final body weight was observed in the 1000 ppm WY groups for both CD and F344 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Localization of the mouse thymidine kinase gene to the distal portion of chromosome 11.

We report the cytogenetic mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary and independent analyses: (1) investigation of chromosome aberrations associated with tk-1 gene inactivation in the L5178Y TK+/- -3.7.2C cell line, and (2) fluorescence in situ molecular hybridization of cloned tk-1 cDNA probes to mitotic chromosomes of this cell line. The consensus location from both analyses is 11E1-E2. Consideration of the mouse tk-1 gene localization, along with evidence that the homologous human TK1 gene is located distally on the large arm of chromosome 17, appears to extend the region of homology between MMU11 and HSA17 to the distal end of both chromosomes.

Animals↗

PubMed1990

Use of DNA purified in situ from cells embedded in agarose plugs for the molecular analysis of tk-/- mutants recovered in the L5178Y tk+/- 3.7.2C mutagen assay system.

We have reported that tk-/- mutants recovered in the mouse L5178Y tk+/- 3.7.2C mutagen assay have often lost the tk+ allele. Allele loss in the tk-/- mutants is documented on Southern blots as the absence of a 6.3-kb Nco I fragment seen in both tk+/+ and tk+/- cell DNAs. For the routine screening of large- and small-colony tk-/- mutants DNAs for the absence of this genomic fragment, we have found that cells can be lysed in agarose plugs, and DNA of cells embedded in plugs can be purified, restricted with Nco I, electrophoresed, and analyzed on Southern blots without significant band distortion or diffusional loss of tk- specific fragments in the 2-7-kb range. Purification and restriction analysis of DNA in agarose plugs, originally developed to allow pulsed-field gel electrophoresis of very large DNA fragments, represents a convenient alternative to conventional DNA purification methods, allowing quantitative recovery of DNA from small numbers of cells, eliminating centrifugation, phenol extraction, and ethanol precipitation steps, and requiring smaller quantities of reagents.

Alleles↗

PubMed1990

Molecular aspects of chemical mutagenesis in L5178Y/tk +/- mouse lymphoma cells.

Southern blot analyses were performed on DNA from at least 10 large and 10 small colony thymidine kinase-deficient (tk -/-) mutants induced by each of 10 mutagens [2-amino-N6-hydroxyadenine (AHA), ethyl methanesulfonate (EMS), methyl methanesulfonate, 2-acetylaminofluorene, methotrexate, caffeine, methapyrilene, 4-(9-acridinylamino)-methanesulfo-m-anisidide, hycanthone methanesulfonate and procarbazine]. Two molecular mutant genotypes were recognized upon digestion with NcoI and subsequent probing with a 1.1 kb cDNA insert from plasmid pMtk 4: (i) no detectable alteration, and (ii) the absence of the functional tkb allele as indicated by the absence of the 6.3 kb fragment. In combination with the previously established chromosomal nature of most small colony tk -/- mutants, this permitted the classification of these 10 mutagens according to the relative proportions of each of four classes of genetic damage they induced. AHA and EMS gave mutational spectra consistent with their point mutational effects in other systems. The other eight mutagens induced mostly small colony mutants, most of which had lost the entire original tkb allele. Methotrexate induced high frequencies of large colony mutants at the tk locus, most of which lacked the tkb allele, although it is weakly or non-mutagenic at the hemizygous hprt locus in these same cells. At least three of these mutagens-methotrexate, caffeine, methapyrilene (and possibly procarbazine)--lack structural alerts for DNA reactivity, implying a major class of non-DNA primary targets for mutagenicity in mammalian cells that interact secondarily with the chromosome. These results are discussed in relation to the known differences in sensitivity among various short-term tests for genotoxicity.

Animals↗

PubMed1981