Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor.

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

Modifications of the antioxidant enzymes in relation to chromosome imbalances in human melanoma cell lines.

Five human melanoma cell lines were investigated for their antioxidant activities. These metabolic data were correlated with cytogenetic analysis giving the relative numbers of chromosomes or chromosomal segments carrying the gene encoding for each enzyme. Particular attention was focused on the expression of superoxide dismutase 2 (SOD2), whose gene, located on the long arm of chromosome 6 (6q), has been proposed as a tumour suppressor gene. The activity of glutathione peroxidase (GPX), glutathione reductase (GSR) and catalase appeared to be unrelated to the relative number of 3q, 8p and 11p arms which, respectively, carry their encoding genes. GPX activity paralleled that of total SOD activity, and GSR variations followed those of GPX, suggesting possible metabolic regulation. Both the activity and the amount of SOD1 immunoreactive protein correlated with the number of chromosomes 21, suggesting a gene dosage effect. The three cell lines with deletions of the 6q arm had lower SOD2 activity and less immunoreactive protein than the two cell lines without 6q deletion. In addition, they demonstrated high thymidine kinase and thymidylate synthetase activities, which are directly linked to the cell proliferation rate. These results strengthen the hypothesis that SOD2 has a function as a tumour suppressor gene, but also suggest that the expression of other antioxidant enzymes might be altered in human melanomas.

Strong decrease in biotin content may correlate with metabolic alterations in colorectal adenocarcinoma.

Short-chain fatty acids are an important source of energy for colonocytes. One of these is propionate, which is metabolized through carboxylation by propionyl-CoA carboxylase (PCC), an enzyme encoded by 2 genes, PCCA and PCCB. The co-factor of this reaction is biotin, a product of intestinal bacterial metabolism, as is propionate. Despite detailed knowledge about the metabolic effects and physiology of biotin, the relative amounts of this vitamin in normal colonic mucosae and in tumour tissue remains quite unknown. The biotin content in normal and cancerous cells from the distal digestive tract was examined on 10 pairs of tissue specimens of colorectal cancer and adjacent normal mucosae using reflectance in situ hybridization (RISH). Having observed a high biotin content in colon mucosae and a low content in colorectal-cancer cells, we then studied the transcription levels of PCCA and PCCB genes in 9 colorectal cancers and the corresponding mucosae. In all cases, the levels of mRNA were lower in colorectal cancers than in normal mucosae, the decrease being always more marked for PCCB than for PCCA. In normal mucosae and in adenocarcinoma cancer cells, PCCA and PCCB transcription levels were strongly related to the amount of biotin detected, but not to the number of chromosomes 13 (which carries PCCA) or 3 (which carries PCCB).

Suppression of apoptosis by overexpression of Bcl-2 or Bcl-xL promotes survival and mutagenesis after oxidative damage.

Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, and its close relative Bcl-xL, are both effective inhibitors of apoptosis induced by a wide variety of stimuli in many different cell types. In a previous study, we reported that suppression of apoptosis by Bcl-2 or Bcl-xL, markedly elevates the levels of radiation-induced mutations at the specific locus thymidine kinase. We investigated the effect of the Bcl-2 or Bcl-xL overproduction on hydrogen peroxide-induced mutagenesis. Oxidative DNA damage has been implicated in biological processes such as mutagenesis, carcinogenesis and aging. Overexpression of either Bcl-2 or Bcl-xL enhances oxidative stress mutagenesis in cells with wild type p53 as well as with mutated p53 protein. These results support the hypothesis that apoptosis plays a crucial role in maintaining genomic integrity by selectively eliminating highly mutated cells from the population.

Suppression of apoptosis by Bcl-2 or Bcl-xL promotes susceptibility to mutagenesis.

Bcl-2 appears to contribute to neoplasia primarily by promoting cell survival, rather than by stimulating cellular proliferation. Bcl-2, and the related protein Bcl-xL, each suppress apoptosis induced by a wide variety of stimuli in many different cell types. Here we report that suppression of apoptosis by Bcl-2 or Bcl-xL markedly elevates the levels of radiation-induced mutations. This enhanced mutagenesis is the result of an increase in mutation frequency (mutations per survivor) together with a moderate increase in viability. Ectopic expression of either Bcl-2 or Bcl-xL enhances radiation mutagenesis in cells with wtp53. Surprisingly, we found that ectopic expression of Bcl-xL also promotes mutagenesis in p53- cells. These results support the hypothesis that apoptosis plays a crucial role in maintaining genomic integrity by selectively eliminating highly mutated cells from the population.