Cell Biochem Funct. 1995 Jun;13(2):99-104. Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro. Carraro U, Bruson A, Catani C, Dalla Libera L, Massimino ML, Rizzi C, Rossini K, Sandri M, Cantini M. Source University of Padova, CNR Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, Italy. Abstract Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate. PMID: 7538914 [PubMed - indexed for MEDLINE]
Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.
CARRARO, UGO;ROSSINI, KATIA;SANDRI, MARCO;CANTINI, MARCELLO
1995
Abstract
Cell Biochem Funct. 1995 Jun;13(2):99-104. Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro. Carraro U, Bruson A, Catani C, Dalla Libera L, Massimino ML, Rizzi C, Rossini K, Sandri M, Cantini M. Source University of Padova, CNR Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, Italy. Abstract Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate. PMID: 7538914 [PubMed - indexed for MEDLINE]
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