Combination of osteoinductive bone proteins differentiates mesenchymal C3H/10T1/2 cells specifically to the cartilage lineage.

During embryonic development, cartilage formation involves the condensation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The embryonic, murine, mesenchymal stem cell line, C3H/10T1/2, is pluripotent; exposure to azacytidine or to bone morphogenetic protein-2 or -4 results in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for 10T1/2 differentiation specifically to the cartilage lineage and at high yields.

The farnesyl protein transferase inhibitor BZA-5B blocks farnesylation of nuclear lamins and p21ras but does not affect their function or localization.

BZA-5B is a peptidomimetic inhibitor of protein farnesylation in mammalian cells. We have examined the specificity of this compound toward inhibition of farnesylation of p21ras and the nuclear lamin proteins, prelamin A and lamin B. We have also used the Raney nickel cleavage technique in conjunction with radio-gas liquid chromatography to assess the ability of this compound to block total protein farnesylation.

Expression of prelamin A but not mature lamin A confers sensitivity of DNA biosynthesis to lovastatin on F9 teratocarcinoma cells.

The role of inhibition of prelamin A processing in the inhibition of DNA synthesis by lovastatin was examined by expressing prelamin A in F9 teratocarcinoma cells. These cells, normally lacking expression of the A/C lamins, were transfected with constructs expressing either prelamin A or mature lamin A and the effect of lovastatin on DNA biosynthesis was assessed. It was found that expression of prelamin A specifically conferred sensitivity to inhibition of DNA biosynthesis by lovastatin on F9 cells.

An antibody which specifically recognizes prelamin A but not mature lamin A: application to detection of blocks in farnesylation-dependent protein processing.

A polyclonal antibody [anti-prelamin A antibody (alpha-PA)] has been obtained against the peptide LLGNSSPRTQSPQN which is proteolytically removed during the farnesylation-dependent processing of prelamin A to mature lamin A. We tested the ability of this antibody to detect inhibition of farnesylation-dependent protein processing of prelamin A. The alpha-PA antibody was shown to immunoprecipitate prelamin A from lovastatin-treated HeLa cells but not mature lamin A from untreated cells.

The processing pathway of prelamin A.

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins.