DNA-Triplex-forming-oligonucleotides as a tool to target specific DNA sequences

Purpose: Triplex-forming-oligonucleotides (TFOs) are able to bind complementary DNA sequences in a sequence specific manner and are therefore a promising tool to manipulate genes or gene regulatory units in a cellular environment. TFOs might have also therapeutic potential e.g. as a carrier for Auger-Electron-Emitter (AEE) to target DNA of tumour cells. A main obstacle is the access of the TFOs to their targets in the cell nucleus. Thus we studied the intracellular biokinetics of TFOs with the focus on the intracellular transfer from the cytoplasm into the nucleus. Method: TFOs specific for the genes p16ink4a and survivin were designed using (TFO Target Sequence Search, Univ. of Texas). DNA-Triplex-formation was confirmed by electrophoretic-mobility-shift-assay (EMSA) in vitro. For biokinetic studies SCL-II cells were transfected by electroporation with Alexa488-labeled TFOs. Transfected cells where subsequently cultured for 1 h, 6 h, 12 h, 18 h, 24 h and 30 h and TFO signal intensity were determined in single cells and in isolated cell nuclei by flowcytometry (FACS-Canto II, BD) at each time point. Results: Sequence design of TFOs by (TFO Target Sequence Search, Univ. of Texas) for the desired genes is generally not suitable to predict DNA-Triplex-formation in vitro as could be demonstrated by EMSA. The desired Triplex-DNA-formation could be confirmed for only two TFOs by EMSA. The biogenetic studies showed that TFO-Alexa488 positive cells were detectable as soon as 1 h after transfection and the signal intensity remained constant for at least 30 h. TFO-positive cell nuclei were not detectable for up to 6 h. After 12 h a significant increase of TFO-Alexa488-positive cell nuclei was observed. Conclusion: Stable Triplex-DNA-formation in vitro can not be predicted by the sequence of TFOs only. TFOs initially located in the cytoplasm are re-located to the cell nucleus within 12 h after delivery of the TFOs probably during cell division

High expression levels of survivin protein does not abolish UV-induced apoptosis in SCL-II cells

Survivin is a member of the inhibitors of apoptosis and has been implicated in both the regulation of cell division and the suppression of apoptosis. Whereas over-expression of survivin correlates with an unfavourable prognosis in many malignant tumours, siRNA against survivin causes a reduction of cell proliferation, the induction of apoptosis and an enhanced radiosensitivity of squamous cell carcinoma cell lines and other tumour cell lines. The apoptotic response and the survivin protein expression levels in the squamous cell carcinoma derived SCL-II cell line were analyzed after exposure to ionizing radiation (IR) and UV radiation. Apoptosis was assessed by PARP cleavage and by flow cytometry (Annexin V-FITC/ PI assay). SCL-II showed a low induction of apoptosis after exposure to IR but a strong apoptotic response after exposure to UV (25% apoptotic cells 5 h after UV), which correlated with PARP cleavage and characteristic changes in cell morphology like membrane blebbing and formation of apoptotic bodies. Cell cycle analysis revealed that exposure to UV, but not IR, induced a G1 checkpoint arrest in SCL-II cells.There was no change in survivin protein expression after exposure to IR and UV compared to controls. One of the used survivin antibodies detected a subband (28 kDa) which correlated with the apoptotic state of SCL-II cells.Conclusions: Survivin expression does not interfere with apoptosis induction in SCL-II cells. UV triggeres apoptosis in SCL-II cells is not abolished by a high expression level of survivin. Apoptosis in SCL-II cells seems to be associated with cell cycle perturbations