Colette J. Rudd

Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control cultures

Mutagenesis assays at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells frequently yield mutant colonies with a bimodal size distribution. The objectives of this study were to determine whether a relationship exists between mutant colony size and chromosomal aberrations and whether the colony-size distributions obtained from this assay can indicate the clastogenic activity of a test chemical. Cells from 8 different types of L5178Y mouse lymphoma cell colonies were examined for chromosomal abnormalities within 10 cell generations after colony isolation. The colonies included small (sigma) and large (lambda) unselected cell (UC) and trifluorothymidine-resistant (TFTr) colonies derived from TK +/- cell cultures treated with the solvent dimethyl sulfoxide (DMSO) or hycanthone methanesulfonate (HYC). Chromosome abnormalities were present in cells from 12% (7/60) of the UC colonies, but there was no apparent relationship between colony diameter and the presence of chromosomal abnormalities. Abnormalities affecting chromosome 11, which is believed to be the site of the TK gene, were not observed in cells from UC colonies. Abnormalities affecting chromosome 11 were observed only in cells from sigma-TFTr colonies irrespective of whether they were spontaneous (5/15 colonies) or induced by HYC (4/15 colonies). Overall, 30% (9/30) of sigma-TFTr colonies had cells with an abnormal chromosome 11 and 10% (3/30) had abnormalities affecting other chromosomes. Abnormalities affecting chromosome 11 were not observed in cells from lambda-TFTr colonies (0/30 colonies). The observation of only 30% of sigma-TFTr colonies with chromosome damage affecting chromosome 11 indicates that other mechanisms, in addition to chromosome damage at the level of resolution used in this study (i.e., 200-300 chromosome bands). contribute to small TFTr colony size.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories. II, Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells

Laboratory protocols and guidelines have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 cells exist, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. The protocols were based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North-American and European governmental, university and contract laboratories involved with in vitro mutation testing. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.

Stable dicentric chromosomes induced by chemical mutagens in L5178Y mouse lymphoma cells

Stable, tandem dicentric chromosomes were discovered in two mutant cell colonies resulting from exposure of L5178Y mouse lymphoma cells to chemical mutagens. These unusual dicentrics were present in all metaphase cells examined from these colonies, even after approximately 65 cell generations in culture. Observation of cells in metaphase and anaphase suggests that the interstitial centromere in these dicentrics is non-functional, and that the terminal centromere is solely responsible for their orderly anaphase segregation.

Correlation of the ability of retinoids to inhibit promoter-induced anchorage-independent growth of JB6 mouse epidermal cells with their activation of retinoic acid receptor γ

Retinoids inhibit the biological effects induced in mouse epidermal cells by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Specific nuclear retinoic acid receptors (RARs) have been identified in the epidermis, but the specific receptor that mediates the inhibitory response by retinoids is not established. Retinoic acid and six conformationally restricted retinoids were evaluated in an in vitro bioassay using the JB6 mouse epidermal cell line. These activities were then compared with the ability of these retinoids to activate the RARs in transient transfection assays for transcriptional activation to identify the retinoid receptor involved in inhibiting TPA-induced anchorage-independent growth. The retinoids inhibited TPA-induced colony formation of JB6 cells in semisolid medium at concentrations that were not toxic based on colony formation of attached cells. These concentrations ranged from less than 10(-9)-10(-6) M. 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylanthracen-2-yl)benzoic acid (TTAB) was the most potent retinoid, with an EC50 of 0.8 nM. Both RAR alpha and RAR gamma were expressed in JB6 cells. Expression of RAR beta was not detected in these cells using a polymerase chain reaction assay, consistent with its extremely low level in mouse skin. Inhibition of the TPA response by these retinoids in JB6 cells correlated only with their transcriptional activation of RAR alpha, but not with that of RAR alpha. These results suggest that RAR gamma is most probably the receptor that mediates the chemopreventive effects of retinoids in mouse epidermis.