In vitro activity: UT-SCC-12A and UT-SCC-12B are both more resistant to Bleomycin sulfate with IC50 of 14.2 nM and 13 nM, respectively. Alveolar macrophages incubated with 0.01 μg/mL to 1μg /mL Bleomycin sulfate for 18 hours secretes significantly more fibroblast growth factor than macrophages incubated without Bleomycin sulfate. Macrophages stimulated with Bleomycin sulfate continues to produce significant amounts of fibroblast growth factor even after Bleomycin sulfate is removed and replaced with fresh (Bleomycin sulfate-free) media. Fibroblast growth factor secretion by Bleomycin sulfate-stimulated alveolar macrophages is inhibited by cycloheximide, and the 5-lipoxygenase inhibitors NDGA (nordihydroguaiaretic acid) and BW755c, indicating not only a requirement for protein synthesis but also for metabolites of the 5-lipoxygenase pathway of arachidonic acid metabolism for full expression of activity. Bleomycin sulfate (400 μg/mL) incubation for 24 hours decreases the viability of NTera-2 cells, and increases caspase-3, -8 and -9 activities, Bax and cytoplasmic cytochrome c levels and decreases Bcl-2 levels. In terms of unstable aberrations, the clastogenic effect of Bleomycin sulfate on ADIPO-P2 cells persists for at least 10 days after exposure. Bleomycin sulfate-induced telomere instability in mammalian cells persists for several generations after exposure. Moreover, the appearance of telomere fusions in Bleomycin sulfate-exposed cells 10 days after treatment suggests that Bleomycin sulfate can induce delayed telomere instability.

Cell Assay: ADIPO-P2 cells are grown in D-MEM high glucose medium supplemented with 20% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C and 5% CO2 atmosphere. Cells are cultured as monolayer in TC25 Corning flasks containing 1.5 × 105 cells/mL. For each experiment, two flasks are set up, one for the control and one for the treated culture. During the log phase of growth ADIPO-P2 cells are treated with a 30 minutes pulse of 2.5 μg/mL of Bleomycin sulfate. Control cultures are set up in parallel but not exposed to Bleomycin sulfate. Time of exposure and concentration of Bleomycin sulfate are chosen according to previous studies carried out in our laboratory with mammalian cells exposed to Bleomycin sulfate. At the end of the pulse treatment with Bleomycin sulfate, the cells are washed twice with Hank's balanced salt solution and kept in culture with fresh culture medium until harvesting. Cells are continuously maintained in culture during 5 passages or subcultures after treatment. Subcultivation is carried out whenever the cultures became confluent (approximately 4 × 105 cells/mL of culture medium). To estimate cell growth, at the time of subcultivation cells are collected by trypsinization, an aliquot of about 200 μL stained with 0.4% trypan blue, and the number of viable cells is determined. Cells are then suspended in fresh culture medium and dispensed into new culture flasks containing 1 × 105 cells/mL to continue growing. The rest of the cells is discarded or dispensed in another flask for cytogenetic analysis, which is performed at 18 hours and 10 days after the end of treatments. To analyze chromosomal aberrations, colchicine (0.1 μg/mL) is added to cell cultures during the last 3 hours of culture. Chromosome preparations are made following standard procedures. After harvesting, cells are hypotonically shocked, fixed in methanol:acetic acid (3:1), spread onto glass slides and processed for PNA-FISH. Two independent experiments are carried out.

Acta Otolaryngol Suppl. 1997;529:241-4; Am J Pathol. 1989 Feb;134(2):355-63; Stem Cell Res Ther. 2012 May 29;3(3):21.