Antisense suppression of p107 inhibits 3T3-L1 adipocyte differentiation

The murine 3T3-L1 preadipocyte cell line is well characterized for its capa city to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene fam ily has been shown to be dramatically upregulated during the early requisit e clonal expansion phase of 3T3-L1 adipogenesis; however, a functional cons equence has yet to be described. A phosphorothioate antisense RNA approach was utilized to determine if inhibition of p107 expression would block or p erturb adipocyte differentiation. A series of three phosphorothioate oligon ucleotides in antisense orientation was generated, designated AS1, AS2, and AS3 along with a sense control oligonucleotide complementary to AS1 and ad ded to postconfluent cells at a concentration of 20 and 50 muM throughout h ormonally stimulated differentiation. Treatment of cells with either concen tration of the sense, AS1, AS2, or 20 muM AS3 oligonucleotides had little e ffect on either Oil Red O lipid accumulation or induction of p107 protein l evels. In contrast, treatment with 50 muM AS3 inhibited the increase in p10 7 protein levels and led to a complete block in differentiation as detected by Oil Red O lipid accumulation and inhibition of adipocyte-specific mRNA expression. In addition, treatment with AS3 led to a significant inhibition of cellular proliferation associated with clonal expansion. Combined, thes e results provide strong evidence supporting a functional role for p107 in 3T3-L1 adipocyte differentiation. (C) 2001 Academic Press.