Phosphorylation of chromosomal proteins from mammary cell lines.

Phosphorylation of histone and none-histone chromosomal proteins in two clonal mouse mammary cell lines having low (V-14) and high (T-19) tumorigenicity was investigated. Logarithmic phase cells were incubated in medium containing 125 muCi/ml[32P]-orthophosphate. Cell nuclei were isolated, chromosomal proteins (histones and non-histones) extracted and their amino acid composition, protein/DNA mass ratios, 32P-uptake and gel electrophoretic patterns examined. Significant differences in the histone and NHC-protein phosphorylation of these two cell lines were found. Comparison of radioactivity profiles of electrophoretically separated T-19 and V-14 NHC-proteins showed that 60% of the T-19 bands in the mol. wt range of 160,000 to 15,000 daltons had increased [32P]-orthophosphate uptake. Differences in histone radiolabelling between V-14 and T-19 were confined to the H1, H3 and H4 classes. [32P]-ATP-pool measurements of V-14 and T-19 at 30, 60 and 120 min of incubation were similar, hence the difference in 32P-uptake was not due to ATP-pool fluctuations. Results of studies on growth rate, growth potential and the possibility that comparisons were made between cells at different stages of the growth cycle indicated that these variables did not account for the higher phosphorylation of T-19 chromosomal proteins. The increased [32P]-phosphate uptake into T-19 cell protein was correlated with an elevated content of NHC and histone proteins and pointed towards a correlation between the degree of phosphorylation and the high tumorigenicity of this cell line.

Effect of calcitonin on the phosphorylation of non-histones from cultured bone cells.

32P-uptake into non-histones from bone cell cultures was selectively stimulated in the presence of calcitonin. Comparison of the control and experimental radioactivity profiles of non-histones fractionated by SDS gel electrophoresis showed that, in response to calcitonin stimulation, there was a 2- to 3-fold increase in the specific activity associated with non-histone proteins in the molecular weight range of 10,000 to 45,000 daltons while that of bands between 50,000 to 200,000 decreased.

Effect of metabolic inhibitors on bone lipid synthesis from radioactive bases.

Iodoacetate at 10(-3) M and 10(-4) M concentrations was found to strongly inhibit incorporation of both 14C-choline and 3H-inositol into phospholipids of calvaria from newborn rats; it had somewhat less effect on ethanolamine incorporation and almost no effect on serine incorporation. Similar though less dramatic results were observed with cyanide or dinitrophenol at similar concentrations.

Effect of beta-aminopropionitrile on in vitro bone lipid synthesis.

The effect of beta-aminopropionitrile (BAPN) on lipid synthesis and 14CO2 production from 14C-acetate by rat calvariae in vitro was investigated by biochemical, histochemical, and autoradiographic techniques. Exposure of bones to 5 mM BAPN throughout a 5-hour incubation period resulted in little effect on lipid synthesis when compared with control bones. Higher BAPN concentrations, up to 100 mM, during this short incubation period resulted in enhanced lipid radioactivity for most of the identified lipid classes. Longer preincubation periods of 20 hours and low BAPN (20 mM) concentrations resulted in enhanced lipogenesis. As BAPN concentrations above 20 mM were employed, lipogenesis decreased to such a level that some lipid classes failed to become labeled with 14C-acetate. 14CO2 production from 14C-acetate by a rat calvariae was affected by BAPN concentration and incubation time in a pattern similar to that of lipid synthesis. Histochemistry demonstrated neutral fat in the osteogenic cells and phospholipids in osteoblasts and the osteoid matrix. Autoradiographic localization of 14C-acetate revealed that the radioactivity was in the osteogenic cells and that this activity was markedly increased after pretreatment of the calvariae with BAPN. This study shows that some concentrations of BAPN enhance lipogenesis by rat calvariae and supports the earlier suggestion that lipids play a role in biologic calcification.