Cytotoxicity of etoposide in cancer cell lines in vitro after BCL-2 and C-RAF gene silencing with antisense oligonucleotides.

BCL-2 and C-RAF genes are overexpressed in most types of cancers. Although these genes are mediators in different molecular pathways their main characteristic is the antiapoptotic activity, thus cells that overexpress either BCL-2 or C-RAF lose their ability to undergo apoptotic death being resistant to chemotherapeutic agents and/or physiologic mediators of cell death (e.g., TNF-alpha). Both anti-C-RAF, and anti-BCL-2 oligonucleotides were tested as chemosensitizers in cancer therapy. The aim of the study was to investigate the effects of the combined use of antisense oligonucleotides (ASOs) targeting BCL-2 and C-RAF transcripts on the in vitro cancer cell cultures exposed to etoposide. Cells were transfected with phosphorothioate BCL-2 and C-RAF ASOs. To sustain high intracellular level of ASOs, 3-day transfection was used, and it was followed by a single treatment with 20 microM etoposide for 5 h. The following cancer cell lines were tested: A549, HeLa, and T24. Sequence-specific decrease in BCL-2, and C-RAF mRNA levels were confirmed by real-time RT-PCR: after 1-day treatment mRNA levels decreased by 9-42% of the normal expression in cells treated with 50-1200 nM ASOs. Also, the induction of cell death in all transfected cultures in a concentration-dependent manner was confirmed by MTT assay,microscopic analysis of cell morphology, and the measurement of histone H3 expression. Results also showed that both ASOs effectively potentiated etoposide-induced cytotoxicity; the strongest effects were obtained in A549 (lung cancer). This observation suggests that lower concentrations of both antisense oligonucleotides may be used, at least for this type of cancer, to obtain high efficiency of etoposide-induced cell death enhancement. Simultaneous use of two ASOs in 3-day treatment allows us to lower concentrations needed to obtain significant treatment results thus enabling to diminish sequence-unspecific toxicity.

Repetitive extragenic palindromic PCR (REP-PCR) as an alternative method for detection of bulking in activated sludge.

Bulking of activated sludge is a world-wide problem which negatively affects wastewater treatment efficiency. The most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria (filamentous bulking) or excess of biopolymers on the surface of non-filamentous microbes (non-filamentous or Zoogleal bulking). Because of the complex nature of the bulking phenomenon finding a successful bulking control strategy remains a very important issue that awaits new options and advices. The REP-PCR fingerprinting method has been applied to distinguish a bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns were compared with each other in terms of the presence or absence of bands and in terms of measured integrated optical density (IOD) of the bands. The obtained fingerprinting patterns, using Ward's clustering method, have been analyzed to determine homology/similarity relations between specific non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing which generally is done based on physicochemical sludge analysis. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be a simple, rapid and effective method revealing differences between populations in non-bulking and bulking activated sludge. It may be useful for routine activated sludge monitoring and may be helpful in the early detection of the bulking process.