Transfection of normal primary human skeletal myoblasts with p21 and p57 antisense oligonucleotides to improve their proliferation: a first step towards an alternative molecular therapy approach of Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD), caused by the absence of dystrophin, is associated with decreased muscle cell proliferation. An increased p21 mRNA level in DMD patients may be involved in the process. In this context we are interested to improve the proliferation of primary human skeletal muscle cells (SkMC) by a reduction in the cell cycle proteins p21 and p57 using the appropriate antisense oligonucleotides (ASO). Therefore a transfection procedure needs to be optimized in which the oligonucleotide enters the SkMC with a minimal loss of cell vitality and high efficiency. Three different formulations, Effectene, DAC40, and SuperFect, were compared. Proliferation was analyzed comparing cells transfected with p21 and/or p57 ASO vs. cells transfected with scrambled ASO using a bromodeoxyuridine assay. Under optimal conditions (a mixture of 0.25 microg ASO, 5 microl Effectene, 0.8 microl enhancer) SkMC transfected with p21 ASO reveal an average increase in cell proliferation of 32.5+/-11% after 24 h. p57 ASO shows the same effect, but concomitant transfection of p21 and p57 does not enhance it. A cell vitality of 78+/-14% after 24 h was determined by the MTT test. SkMC transfected with DAC40 reveal a maximal increase in proliferation of 38+/-7% after 48 h and show a vitality of 65+/-8%. In contrast to both these formulations, SuperFect was found to be highly toxic for SkMC, with more than 70% dead cells after 24 h. The increase in proliferation, the functional biological effect of p21 ASO, is well correlated with a decrease in p21 detected by western blot analysis of 31.6% for Effectene. Transfection efficiency was measured directly by FACS analysis using FITC-labeled ASO and data showing ASO internalization in 75.8+/-11.2% of the cell population for Effectene and 74.4+/-6.6% cells for DAC40. Taken together transient transfection of p21 or p57 ASO into primary human SkMC using Effectene significantly improves their proliferation compared to transfection with scrambled ASO without a major loss of cell vitality. This represents a basis for the transfer of this technique to dystrophin-deficient SkMC cultures and the introduction of the short interference-RNA technique which might enhance the effect on cell proliferation.
Department of Biotechnology, University of Applied Sciences, Seestrasse 64, 13347 Berlin, Germany.
Stefanie
S
Endesfelder
Sabine
S
Bucher
Alexander
A
Kliche
Regina
R
Reszka
Astrid
A
Speer
D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050761:Cyclin-Dependent Kinase Inhibitor p57; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D018101:Mice, Inbred mdx; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C493956:CDKN1A protein, human; C493964:CDKN1C protein, human; C493957:Cdkn1a protein, mouse; C493965:Cdkn1c protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050761:Cyclin-Dependent Kinase Inhibitor p57; D016213:Cyclins; D016189:Dystrophin; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger