Materialsand Methods

2.1. DES-Induced Pituitary Tumors in Rats

Four week old male Wistar rats were submitted to subcutaneous implantation of silastic capsules containing DES (10 mg/capsule). Four months later the animals were

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divided into experimental groups (each of 7 rats) receiving daily evening injections of MLT (25 (ig), CGP 52608 (25 (g) or solvent (controls), during 10 days. Two hours before the end of experiment the animals were injected intraperitoneally with bromod-eoxyuridine (BrDU, 50 mg/kg b.w.). The incorporated BrDU was revealed by the immunocytochemical method and the number of BrDU-positive cell nuclei/1000 randomly scored pituitary cells was used as an index of cell proliferation.

2.2. Colon 38 Tumors in Vivo

Male mice, first generation of the cross-bred between C57BL/6 and DBA/2 strains were used in the experiment. The animals were subcutaneously implanted with Colon 38 cells suspension. Ten days after tumor implantation, the animals were divided into six experimental groups, and then received subcutaneously the following substances in the daily evening injections: MLT, 10 (g or 100 (g, CGP5 2608 10 (g or 100 (g, saline or 10% ethanol in saline, during 6 days. The animals were sacrificed by spinal cord dislocation 12 hours after the last injection. Two hours before the sacrifice all the animals were injected i.p. with BrDU in a dose as above. The assessment of cell proliferation was done as in the previous experiment.

2.3. Colon 38 Cells in Vitro

The tumor cells were isolated from Colon 38 tumors and cultured according the procedure described in details elsewhere (5). In brief, the tumor cells were incubated 24 hours in RPMI medium supplemented with 15% fetal calf serum in the presence of [3H]thymidine and in the presence or absence of the investigated compounds. The incorporation of [3H]thymidine into DNA was considered as an index of cell proliferation.

2.4. StatisticalAnalysis

The statistical analysis of the data was performed using the Mann-Whitney test.

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