Biomedical subjects

A Mahonen

Publications and source records attributed to A Mahonen.

25 records · Page 2Linked to original sources

PubMed1990

Affinity of MC 903 for 1,25-dihydroxyvitamin D receptor and its effects on the synthesis of osteocalcin in human osteosarcoma cells.

MC 903 is a new structural analog of the naturally occurring, biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. MC 903 and 1,25(OH)2D3 have shown similar receptor binding properties and comparable effects on leukemic cell differentiation. However, MC 903 is at least 100 times less potent in influencing calcium metabolism than 1,25(OH)2D3. We have therefore studied, how MC 903 competes for the binding sites of 1,25(OH)2D3, influences the 1,25(OH)2D3 induced synthesis of the most abundant bone non-collagenous protein, osteocalcin, and induces the activity of alkaline phosphatase in MG-63 human osteosarcoma cells. We found that the new compound binds to 1,25(OH)2D3 receptors and regulates receptor mRNA levels essentially like the natural ligand. Our results also indicate that MC 903 induces the synthesis of osteocalcin and the activity of alkaline phosphatase in MG-63 cells through a receptor-mediated process almost identically with 1,25(OH)2D3. Growth of the MG-63 cells was inhibited slightly more with MC 903 than with 1,25(OH)2D3.

Alkaline Phosphatase↗

PubMed1990

Affinity of 22-oxa-1,25(OH)2D3 for 1,25-dihydroxyvitamin D receptor and its effects on the synthesis of osteocalcin in human osteosarcoma cells.

A new 1,25-dihydroxyvitamin D3 analog, 22-oxa-1,25(OH)2D3, which may have pharmaceutical use, e.g., in the treatment of psoriasis, was studied using cultured MG-63 human osteosarcoma cells. We found that the new compound binds to 1,25-dihydroxyvitamin D receptors and regulates receptor mRNA levels like the natural ligand. Our results also indicate that 22-oxa-1,25(OH)2D3 induces the synthesis of osteocalcin and the activity of alkaline phosphatase in MG-63 cells through a receptor-mediated process identically with 1,25(OH)2D3.

Alkaline Phosphatase↗

PubMed1990

Effect of 1,25(OH)2D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells.

Hormone-dependent accumulation of specific binding sites for 1,25(OH)2D3 and changes in human 1,25-dihydroxy-vitamin D receptor (hVDR) mRNA levels were examined in cell lines (MG-63, SaOs-2 and U2-Os) derived from human bone. Osteocalcin synthesis and secretion as well as alkaline phosphatase activity were also characterized as biochemical markers of the osteoblastic phenotype. Specific binding sites for 1,25(OH)2D3 were quantified by incubating cultured intact cells with [3H]1,25(OH)2D3 at 37 degrees C. Based on the uptake of 1,25(OH)2D3, there were about 3000 to 4000 receptor molecules per cell with apparent dissociation constants varying between 0.02 to 0.03 nM. The binding was saturated with 1,25(OH)2D3 in 3 to 6 h after the hormone addition and further exposure to the hormone resulted in an upregulation of the bindings sites. The levels were elevated by as little as 10 to 200 pM 1,25(OH)2D3, and maximal binding was achieved with 0.2-0.7 nM 1,25(OH)2D3. Treatment with 1,25(OH)2D3 also resulted in a clear increase (about 3-fold) in hVDR mRNA by 24 h in all three cell lines. The increase in hVDR mRNA level was time- and dose-dependent. MG-63 cells responded with 2- and 15-fold increases, respectively, in intracellular and secreted levels of osteocalcin after the 1,25(OH)2D3-treatment. In dot-blot hybridization assay, MG-63 cells expressed osteocalcin mRNA which was inducible with 1,25(OH)2D3 while, in SaOs-2 and U2-Os cells, osteocalcin mRNA was not detected under the same circumstances. Also, no secretion of osteocalcin was detected in SaOs-2 and U2-Os cells with or without addition of 1,25(OH)2D3.

Alkaline Phosphatase↗

PubMed1988

Separation and quantification of histone H1 subtypes and high-mobility-group proteins by reversed-phase liquid chromatography: protein levels in rat tissues during postnatal development.

The rapid separation and quantification of histone H1 subtypes and high-mobility-group (HMG) chromatin proteins by reversed-phase liquid chromatography on a butylsilica-based column is described. The proteins were fractionated by means of a multi-step acetonitrile gradient containing 0.1% trifluoroacetic acid. This system is capable of resolving the four main HMG proteins (1, 2, 14 and 17), HMG I, protein P1 with HMG 18 and HMG 19 (in one peak) and five histone H1 subtypes in a single 33-min analysis. This method was used to study levels of these chromosomal proteins in nuclei of rat liver, spleen, testis and thymus during postnatal development from 1 to 20 weeks of age. Although no clear tissue specificity of the HMG proteins was apparent, there were significant differences in the relative amounts of these proteins in different tissues. The relative amount of HMG 1 increased from 1 to 12 weeks of age and decreased thereafter, whereas those of HMG 14 and HMG 17 remained almost unchanged. Marked quantitative differences were observed in the five histone H1 subtypes in different tissues. The largest changes in their levels during development were found in the liver and the smallest changes in the thymus. The changes in the spleen and testis were intermediate. These results suggest that the changes in the relative amounts of histone H1 subtypes and HMG proteins observed during postnatal development of the rat may result from differences in the structure of chromatin in these tissues and thus reflect the activity of molecular mechanisms involved in replication and differentiation of the cells.

Aging↗

PubMed1988

Selective decrease in low-Mr HMG proteins HMG I and HMG Y during differentiation of mouse teratocarcinoma cells.

We have studied the presence of high-mobility-group (HMG) chromatin proteins in undifferentiated F9 mouse teratocarcinoma cells and F9 cells, which were induced to differentiate by treatment with retinoic acid and dibutyryl-cAMP for 5 days. Acetic acid/urea-polyacrylamide gel electrophoresis and reversed-phase HPLC revealed that the induced F9 cells contained 77 and 62% less HMG I and HMG Y, respectively, than their untreated counterparts. The relative amounts of two other low-Mr HMG proteins HMG 14 and HMG 17 remained essentially unchanged and only a minor decrease was observed in the content of one of the high-Mr HMG proteins, HMG 2. The identity of the low-Mr HMG proteins was verified by amino acid analysis or partial sequencing. These results suggest that HMG I and HMG Y are HMG proteins specific for undifferentiated cells.

Amino Acid Sequence↗

PubMed1987

Phosphorylation of high-mobility-group chromatin proteins by protein kinase C from rat brain.

Chromosomal high-mobility-group (HMG) proteins have been examined as substrates for calcium/phospholipid-dependent protein kinase C. Protein kinase C from rat brain phosphorylated efficiently both HMG 14 and HMG 17 derived from calf thymus and the reactions were calcium/phospholipid-dependent. About 1 mol of 32P was incorporated per mol of HMG 14 and HMG 17. Phosphopeptide mapping suggested that the same major site was phosphorylated in both proteins at serine. The apparent Km values for HMG 14 and HMG 17 were about 5 microM. HMG 14, HMG 17 and the five histone H1 subtypes prepared from rat thymus, liver and spleen were phosphorylated by the kinase. HMG 14 and HMG 17 from transformed human lymphoblasts (Wi-L2) were also phosphorylated in a calcium/phospholipid-dependent manner. HMG 1 and HMG 2 from the tissues examined were found to be poor substrates for the kinase.

Animals↗

PubMedDate not supplied

Vitamin D(3) analogs (MC 1288, KH 1060, EB 1089, GS 1558, and CB 1093): studies on their mechanism of action.

Selected 20-epi and 20-normal vitamin D(3) analogs were studied. First, point mutations were introduced into human vitamin D receptor (VDR) to identify residues important for ligand binding. In helices three, four and five, His229, Asp232, Ser237 and Arg274 seem to have an important role in the binding of calcitriol. Surprisingly, the 20-epi analog MC 1288 did not bind to Ser237. Second, the effects of analogs on VDR degradation were studied. The transcriptionally active 20-epi analogs protected VDR against degradation more efficiently than the 20-normal analogs and calcitriol. With proteasome inhibitor MG-132 formation of Sug-1-RXRbeta-VDR-VDRE complex was detected. The 20-epi analogs effectively prevented its formation. Thus, the 20-epi analogs induce a VDR conformation, which prevents binding of factors mediating VDR degradation. Third, the analogs were found to be powerful regulators of cell cycle progression in MG-63 cells. They arrested cell cycle in the G0/G1 phase at lower concentrations and earlier time points than calcitriol. This was accompanied by hypophosphorylation of Rb followed by strong inhibition of Cdk2 activity. This correlated with increased levels of p27. Cdk2 and cyclin E levels were downregulated but those of p21 and cyclin D1 were not affected. Thus, a similar sequence of events with calcitriol and the analogs in inhibiting MG-63 cell growth was detected but the analogs had much longer lasting and stronger effects than calcitriol. A unifying scheme for the varying effects of vitamin D(3) analogs is presented.

Animals↗