Resistance to Bleomycin in Cancer Cell Lines Is Characterized by Prolonged Doubling Time, Reduced DNA Damage and Evasion of G2/M Arrest and Apoptosis

Abstract

BackgroundTo establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines. ResultsCompared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05). ConclusionBleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.

Preclinical efficacy of the synthetic retinoid ST1926 for treating adult T-cell leukemia/lymphoma

“…26,33,34 We observed a substantial increase in the DNA damage marker, H2AX phosphorylation (g-H2AX), as early as 2 hours in tested cells ( Figure 3D). Some tumor cells show elevated baseline levels of DNA damage 35 as noted in MT-2 cells and to a lower extent in Jurkat and Molt-4 cells (Figure 3D), which may be due to several factors such as chromatin instability 36 and damaged telomeres. 37 Tax has been shown to increase genetic instability by inducing DNA double strand breaks as evidenced by increased g-H2AX levels 38 ; however, other factors may be also implicated as shown by the variability in the expression of endogenous g-H2AX levels in HuT-102 and MT-2 cells.…”

Regulation of tissue factor in NT2 germ cell tumor cells by cisplatin chemotherapy

“…Bleomycin and etoposide are used together with cisplatin in the PEB protocol to treat GCTs and have also been shown to induce G2/M arrest in cancer cells [15,16]. In a proof-of-concept experiment, we treated NT2 cells for 48 hrs with any of the three agents and assessed, in addition to cellular TF PCA, their cytotoxic and cytostatic effects by trypan blue exclusion (reflecting cell viability) and cell count (reflecting G2/ M arrest), respectively.…”

Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycans Are Targeted by Bleomycin in Cancer Cells

“…Resistance to bleomycin and bleomycin-induced lung fibrosis are two major clinical concerns [30]. Fibrosis, a hallmark of clinical diseases that affects the normal functions of lung, liver, heart, or kidney, is defined as an abnormal accumulation of extracellular matrix components.…”

“…Even though bleomycin interrupts DNA replications once inside of cells [3, 29], the IC 50 of bleomycin varies greatly in different cancer cell lines [30]. It turns out that bleomycin does not diffuse through the plasma membrane but gets inside cells through receptor-mediated endocytosis.…”