Visit our Expo - Redox and Inflammation signaling 2012

Session XVII : Cell signaling in health and disease Poster XVII, 58
Impact of Rb knock down on the modulation erufosine signal transduction in human
leukemia cells
Maya M. Zaharieva1,2, Milen Kirilov2, Minqiang Chai2, Stefan Berger3, Spiro M.
Konstantinov1,2, Martin R. Berger2
1Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str, 1000 Sofia, Bulgaria
2German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany
3Central Institute for Mental Help, Department of Molecular Biology, J5, 68 159
Mannheim
The retinoblastoma gene Rb1 is a prototypical tumour supressor. Consistent with its tomoursupressor
function Rb is known to inhibit proliferation. Recent studies indicate its role in
suppressing apoptosis, too. Loss of Rb sensitizes cells to apoptosis, but loss of Rb can only
contribute to tumor development under conditions in which apoptosis response is already
compromised. The new alkylphosphocholine erufosine is an inductor of Rb in leukemic cells.
Therefore, we decided to suppress the expression of RB using conventional phosphorotioate
antisense oligonucleotides and siRNAs. Unfortunately, both types of oligonucleotides used
didn’t cause stable and reproducible knock-down and we initiated viral transfection
experiments for stable Rb knock-down in SKW-3, BV-173 and K-562 cells. The antisense
sequence was firstly cloned in the pSUPER vector and tested on HEK 293T cells. For
normalizing the transfection efficiency an EGFP-expressing vector was used in addition. To
realize the transfer of the H1-shRNA cassette directed to the Rb-mRNA, the cells were
infected with LentiLox virus which co-express an EGFP-protein. The resulting levels of target
mRNA were measured by real-time RT-PCR after excluding of non-fluorescent cells by flow
cytometry and cell sorting. The highest decrease of the Rb-mRNA (about 86%) was found in
SKW-3 cells. A concomitent knock-down at the protein level was evidenced by Western
blotting. The proliferation activity of the infected cells was estimated by colorimetric MTTdye
reduction assay before and after erufosine treatment. Comparison of Rb knock-down and
wild type leukemic cells revealed the presence of significant differences in cell cycle duration
and sensitivity to erufosine, thus confirming the impact of Rb modulation for the mode of
action of alkylphosphocholines which are known as promising antineoplastic signal
transduction modulators.
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