The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation

Abstract

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [ 3 H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells

“…In differentiation assays, all-trans-RA (SIGMA, Saint Louis, MO, USA) was used at the final concentration of 5 mM as previously described. 48 Medium containing RA was changed every 3 days. For stable transfections, LAN-5 cells grown at 70% confluence were transfected with the expression vectors (pMIREV I1, pMIREV I2 and pMIREV I3), which transcribe hairpin RNAs processed in the cells to become micro RNAs and the control pMIREV ILuc, using the calcium phosphate precipitation technique as described previously.…”

“…48 Immunoprecipitation was carried out as follows: 500 mg of cellular extract was preincubated with protein G-Sepharose beads (Pharmacia, Biotec. Uppsala, Sweden) for 45 min at 41C.…”

“…Protein separation on SDS-polyacrylamide gels and Western blot analysis were carried out as described previously. 48 Antibodies used were anti-IGFBP-5 (c-18) from Santa Cruz Biotechnology …”

Cyclin D1-dependent regulation of B-myb activity in early stages of neuroblastoma differentiation

“…Supernatants were separated on SDS ± PAGE and Western blot analysis using specific antibodies was carried out as previously described. 30 Antibodies used were: a-B-myb (sc-725), a-cyclin A (sc-751), acyclin D1 (sc-717), a-Sp1 (sc-59) from Santa Cruz Biotechnology, Inc., a-HSP-70 (SPA-820) Stress Gen Biotechnology Corp., a-HSP-90 (H38220), a-p21 (C24420) BD Transduction Laboratories.…”

“…Protein analyses were performed as follows: (a) lysate was centrifuged at 14 0006g at 48C for 10 min, the Cell Death and Differentiation B-myb activity during early stages of differentiation V Cesi et al supernatant was collected and protein concentration was determined using a colorimetric assay (BioRad) and (b) 100 mg of total extract per lane were separated on SDS ± PAGE and Western blot analysis using specific antibodies was carried out as previously described. 30 Immunoprecipitation was carried out in 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Nonidet P-40 (ICN Biomedical Inc.), Pefablock SC (Roche) and Complete Protease Inhibitor Cocktail (Roche) at the manufacturer's indicated concentrations. Five hundred micrograms of cellular extracts were pre-incubated with Protein A-Sepharose beads (Amersham-Pharmacia Biotech.)…”