Identification of a hTid-1 mutation which sensitizes gliomas to apoptosis.

Human Tid-1 (hTid-1) is a DnaJ chaperone protein with homology to the Drosophila tumor suppressor Tid56. We report the first case of a tumor-associated mutation at the human TID1 locus, which was identified in the SF767 glioma cell line giving rise to aberrantly high levels of a hTid-1(L) mutant variant. In this study, we set out to determine whether this change in hTid-1 status influences the response of glioma cells to adenoviral (Ad)-mediated delivery of the two major isoforms of TID1, hTid-1(L) and hTid-1(S). Ad-hTid-1(S) induced apoptosis in hTid-1 mutant SF767 cells, while causing growth arrest in wild-type hTid-1-expressing U373 and U87 cells. By contrast, Ad-hTid-1(L) infection had no apparent effect on glioma cell growth. The apoptosis induced by hTid-1(S) was accompanied by mitochondrial cytochrome C release and caspase activation and blocked by stable overexpression of Bcl-X(L). Our findings suggest that the status of hTid-1 in gliomas may contribute to their susceptibility to cell death triggers.

Effect of dietary supplementation on the frequency of spontaneous lacZ mutations in the developing colon.

Epidemiological studies have demonstrated that dietary modifications can reduce the incidence of cancer. Specifically, diets high in vegetables and fruits are associated with lower rates of cancer at many sites. Somatic mutations have a critical role in carcinogenesis suggesting the use of in vivo mutation assays as an alternative approach to studying the relationship between diet and cancer. Since the rate of accumulation of spontaneous mutations is highest during growth and development early in life, we tested whether certain foods as dietary supplements could reduce the rate of mutation during this period using lacZ transgenic mice. Pregnant female mice were placed on a control diet or a diet supplemented to 20% final dry weight with broccoli, cabbage, carrots, flaxseed, green peas, green peppers, oranges or strawberries for the entire duration of their pregnancy and lactation. Mutation frequencies were subsequently measured at the lacZ transgene in colonic epithelial cells of the offspring at 3 weeks of age. A small number of measurements were also made on siblings at 8 weeks of age. While the control AIN-96G diet on its own resulted in lower mutant frequencies than had been observed in earlier experiments with lab chow, no significant reduction in mutant frequencies was detected for any of the foods tested as compared to the AIN-93G diet alone. Significantly more mutations were found at 3 weeks of age in mice fed diets supplemented with broccoli or oranges, but the result with oranges may be the result of jackpot mutations.

The influence of dietary flaxseed and other grains, fruits and vegetables on the frequency of spontaneous chromosomal damage in mice.

Spontaneous genetic damage, whether mutations or chromosomal aberrations, undoubtedly arise from a variety of sources including replication errors, oxidative damage, background radiation, and chemical exposure. Given the numerous correlations between diet and cancer, it seemed possible that diet could influence the spontaneous rate of DNA damage and its genetic consequences. Since diets high in vegetables, fruits, and grains are associated with lower rates of cancer, we supplemented the diets of mice and measured the frequency of micronuclei in the peripheral blood. Micronuclei arise from broken chromosomes or chromosome loss in the erythroblast. They are first seen in the short reticulocyte stage of the red blood cell but persist for the entire 30-day lifespan of the cell in mice. C57Bl mice were placed on a defined diet (AIN-93G) supplemented to 20% final dry weight with grains or freeze-dried fruits or vegetables. The micronucleus frequency was measured in a pre-exposure blood sample and every 2 weeks thereafter for 6 weeks. This was possible in spite of the low spontaneous frequency of 1/1000-2/1000 cells by the use of a novel flow cytometric method, which permitted the analysis of both the mature red blood cells and reticulocytes. Of the foods tested, flaxseed proved to be the most protective by reducing the incidence of micronuclei in both the reticulocyte and normochromatic erythrocyte cell populations by 30 and 11%, respectively. The results show that at least one class of spontaneous genetic damage can be modified by diet and suggests that short-term experiments with small numbers of animals can be used to identify dietary anticarcinogens that may influence human cancer rates.

A mouse homologue of the Drosophila tumor suppressor l(2)tid gene defines a novel Ras GTPase-activating protein (RasGAP)-binding protein.

p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1(L) and hTid-1(S) forms of the human TID1 gene that exhibit opposing effects on apoptosis. We demonstrate that both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells.

Effect of maternal folate levels on somatic mutation frequency in the developing colon.

Folic acid deficiency is associated with an increase in chromosomal aberrations in adult rodents and humans. Somatic mutations have a critical role in carcinogenesis. Since most mutations arise during early development, the effect of maternal folic acid levels on the spontaneous mutant frequency in the developing colon was examined using lacZ transgenic mice. No significant difference in mutant frequencies at both 3 and 8 weeks of age were found between offspring whose mothers were fed low folate and those on high folate diets during pregnancy. Our results suggest that the correlation between folic acid intake and cancer risk may only be effective at extreme folate deficiencies or in combination with other dietary deficiencies or an underlying predisposition.