It has been proposed that binding of BLM to DNA occurs via the hydrogen atom of the amide group in the carboxyl amide moiety, which forms a hydrogen bond with the 2-keto group of the thymine

It has been proposed that binding of BLM to DNA occurs via the hydrogen atom of the amide group in the carboxyl amide moiety, which forms a hydrogen bond with the 2-keto group of the thymine. a process that results in strand splitting. Here, using A549 human lung cells or BEAS-2B cells lunc cells, we show that the cell toxicity of BLM can be suppressed by addition of inorganic polyphosphate (polyP), a physiological polymer that accumulates and is released from platelets. BLM at a concentration of 20 g ml?1 causes a decrease in cell viability SKF-86002 (by ~70%), accompanied by an increased DNA damage and chromatin expansion (by amazingly 6-fold). Importantly, the BLM-caused effects on cell growth and DNA integrity are substantially suppressed by polyP. In parallel, the enlargement of the nuclei/chromatin in BLM-treated cells (diameter, 20C25 m) is normalized to ~12 m after co-incubation of the cells with BLM and polyP. A sequential application of the drugs (BLM for 3 days, followed by an exposure to polyP) does not cause this normalization. During co-incubation of BLM with polyP the gene for the is upregulated. It is concluded that by upregulating this enzyme polyP prevents the toxic side effects of BLM. These data might also contribute to an application of BLM in COVID-19 patients, since SKF-86002 polyP inhibits binding of SARS-CoV-2 to cellular ACE2. test was applied. The average values came from ten independent experiments. Values of < 0.05 were considered statistically significant (*). The calculations were performed with the GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). 2.6. DNA Stand Break Assay The Fast Micromethod was applied to determine DNA damage in A549 cells as described [56]. This system bases on the property of the specific fluorochrome dye PicoGreen (#P0990, Sigma) to form a stable complex with double-stranded DNA [57]. Increased breakage in DNA is reflected by a decrease of the fluorimetric signal which is proportionate to an increasing single-strand DNA breakage. The cells are lysed in the microplates in the presence of SDS (sodium dodecyl sulfate) containing PicoGreen. The DNA denaturation kinetics is recorded after addition of an alkaline NaOHCEDTA solution for 20 min at an excitation of 485 nm and an emission of 520 nm in a Fluoroscan II reader (Labsystems, Finland). The results are expressed by the strand scission factor (SSF) which is, for practical reasons, in a reciprocal way. In turn, a SSF (?1) value from 0.1 to 1 1.0 indicates an increase in the extent of damaged DNA. The vales were calibrated towards calf thymus double-stranded DNA (#D4522, Sigma-Aldrich). A549 cells were seeded at a density of 40,000 cells cm?2 in SKF-86002 8-well plates. After an incubation period of 3 days the cells were collected and used for the break determinations. Incubations with BLM, polyP or combinations are specified with the respective experiments. Five parallel assays were performed. The significance was calculated by ANOVA (by Bonferroni adjustment) program [58]. 2.7. Quantitative Real-Time Polymerase Chain Reaction: BLM Hydrolase A detailed description of the qRT-PCR (reverse transcriptase-PCR) procedure applied has been given before [59]. The A549 cells were exposed to polyP, BLM or SKF-86002 the two compounds in co-incubation, or remained untreated (controls); the respective concentrations are given under Results. Cells were Rabbit Polyclonal to RAB41 incubated in 8-well plates (40,000 cells cm?2) and incubated for 1 or 3 days. Then RNA was extracted, reverse-transcribed into cDNA using reverse transcriptase (Promega Corp., Madison, WI, USA) and an oligo(dT)16 primer (Promega). The amplified PCR products were obtained after 28C32 cycles (30 s at 94 C, annealing for 45 s at 50 C, primer extension for 30 s at 72 C). For the amplification of the BLM hydrolase [60,61,62], the forward primer 5-ACCAGCCCATTGACTTCC-3 and the reverse primer 5-TGTCCACCACCACTTCGT-3 were used. The values were normalized with the expression levels of the reference housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase; NM_002046.3) with the primer pair Fwd, 5-ACTTTGTGAAGCTCATTTCC-3.