Transformation by radiation of rat foetal glial cells.

PURPOSE: To establish and characterize an in vitro model of radiation-induced transformation of normal glial cells. MATERIALS AND METHODS: During the last week of gestation, pregnant Sprague-Dawley rats were either irradiated at 3.5 Gy (0.022 Gy h(-1)) with a 60Co source or sham irradiated. On day 21 of gestation, cortical nerve cells from foetuses were isolated, and then maintained in culture for about 100 passages, in presence of 10(-9) g/ml of tetradecanoyl phorbol acetate (TPA). To follow transformation, various parameters: cell type, proliferation, clonogenicity, karyotypes and tumorigenicity, were studied at different passages. RESULTS: As the number of passages increased, control cells lost their glial morphology and were immortalized. They kept on expressing specific markers of type 2 astrocytes (glial fibrillary acid protein (GFAP) and A2B5). Karyotypes remained near diploid. At all passages tested, they were not tumorigenic in nude mice. Irradiated cells expressed the 2A progenitor cell specific markers: GFAP, vimentin and A2B5. Karyotypes evolved toward polyploidy and cells displayed an iso 7 and a marker. These changes were synchronous with modifications in tumorigenicity. Metastases were even observed in nude mice. CONCLUSIONS: Cells from irradiated animals were fully transformed, while cells from sham irradiated animals were only immortalized.

Purine metabolism in two human melanoma cell lines: relation to proliferation and differentiation.

Purine nucleotide metabolism was studied in two human cutaneous melanoma cell lines IPC182 and IGR221. IPC182 cells do not differentiate, while IGR221 cells differentiate spontaneously at confluency, with intense melanin production. The activities of 11 enzymes involved in the de novo or salvage synthesis or the catabolic pathway of purine nucleotides were measured at different times (from day 3 to day 18), after subculture, during exponential growth and the stationary phase, with or without differentiation. The results demonstrated remarkable differences in the enzyme activity levels and/or the evolution from exponential growth to the stationary phase for each cell line, as well as between the two cell lines. In the non-differentiating IPC182 cells, the activity of enzymes involved in purine nucleotide synthesis decreased when the growth rate slowed down and remained at a low level with a concomitant increase in catabolic activities. In the differentiating IGR221 cells, the activity of enzymes involved in purine nucleotide salvage synthesis increased during the proliferative phase and was maintained at a high level when the cells reached confluency and differentiated; catabolic activities were always lower than in the IPC182 cells. This suggests that extra purine nucleotides, synthesized preferentially by the salvage pathway, could be required for the differentiation of human melanoma cells. Since the two cell lines were cultured in the absence of any differentiation-inducing agents, these results indicate that various metabolic modifications are associated with the natural processes of cell proliferation and differentiation. This research could help to identify some of the enzymes involved in purine metabolism as the targets for the induction of differentiation.

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.

[Nucleotide metabolism in human glioma: comparative study of primary tumors, grafted tumors on nude mice and cell cultures]. / Métabolisme des nucléotides dans les gliomes humains: comparaison de tumeurs primaires, de tumeurs implantées sur souris nude et de lignées.

A study developed to test the hypothesis of a possible relationship between metabolic modifications and chromosomal imbalances in solid tumors leads us to investigate the metabolism of purine nucleotides in human gliomas. In order to assess the representativeness of experimental models frequently used, the activities of nine enzymes involved in the synthesis and in the catabolism of purine nucleotides were measured on samples of normal brain, primary and xenografted glial tumors and cell cultures established from human gliomas. In parallel, two enzymes involved in pyrimidine metabolism were also studied on the same samples. The results highlight the low activity of the purine metabolism in human gliomas when compared to normal brain, tissue with low proliferative activity. On the contrary, the pyrimidine metabolism in human gliomas is increased by comparison to normal brain. For the purine metabolism, few differences are observed between enzyme activities calculated in primary glial tumors, xenografts and cells in culture. In grafted tumors and cell cultures, the activity of this metabolism is similar or lower than in normal brain, except for inosine monophosphate dehydrogenase. However, for the pyrimidine metabolism, significantly differences are observed between primary glial tumors, grafted glial tumors and cell cultures. The thymidine kinase/thymidylate synthetase ratio depends on the model studied. These results point out the problem of the representativeness of these models, especially when used for experimental therapeutic studies. This metabolic study also underlines that all results should be interpreted carefully and that the limits for the use of these two experimental models should always be clearly exposed.

Establishment and characterization of human ocular melanoma cell lines.

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.

Use of unstable chromosome aberrations for biological dosimetry after the first postirradiation mitosis.

The loss of unstable chromosome aberrations after the first postirradiation mitosis makes their use difficult in radiation dosimetry. We describe here a method which, in a cell population observed at this stage, allows retrospective estimation of the frequencies of the unstable aberrations induced at the time of irradiation, and their use as a dosimeter. The laws controlling the behavior of unstable aberrations during mitosis were defined from a large-scale experiment on irradiated human lymphocytes. For cells undergoing the first, second, or third mitosis after irradiation, relationships were determined between the frequency, at irradiation time, of acentric fragments not arising from formation of dicentrics or rings, and the ratio of dicentrics and centric rings appearing without acentric fragments to the total number of dicentrics plus rings. On the basis of this ratio, the method described here provides an assessment of the postirradiation mitotic activity in a cell population. This assessment permitted estimation of the cell distribution and frequency of dicentrics plus centric rings, and of the frequency of acentric fragments at the time of irradiation. The use of this method for retrospective dosimetry after whole-body irradiation under various conditions of exposure is illustrated.