Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. (1/960)

p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation.  (+info)

1Alpha,25dihydroxyvitamin D3 and platinum drugs act synergistically to inhibit the growth of prostate cancer cell lines. (2/960)

The majority of men who die from prostate cancer (PC) have hormone-refractory disease. To date, chemotherapeutic agents have had little or no impact on the survival of such patients. To explore a new approach for the treatment of hormone-refractory PC, we examined the combination effects of cis- or carboplatin with vitamin D. 1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its synthetic analogue, Ro 25-6760, have an antiproliferative effect on some prostate cancer cell lines. Consequently, the growth-inhibitory effects of the drugs were measured, both singularly and in combination with cis- or carboplatin, on PC cells. Our results show that although each of the drugs alone displayed antiproliferative activity, the growth inhibition of PC cells was further enhanced by the combination of 1alpha,25(OH)2D3 or Ro 25-6760 and either platinum agent. The greatest enhancement of inhibition occurred using smaller concentrations of the platinum compound in combination with higher concentrations of 1alpha,25(OH)2D3. Isobologram analysis revealed that 1alpha,25(OH)2D3 and platinum acted in a synergistic manner to inhibit the growth of PC cells. Our findings suggest that there is potential clinical value in combining 1alpha,25(OH)2D3 with platinum compounds for the treatment of advanced-stage human PC.  (+info)

Plasma 25-hydroxyvitamin D in growing kittens is related to dietary intake of cholecalciferol. (3/960)

Vitamin D synthesis by growing kittens exposed to ultraviolet light is ineffective. Concentration of 25-hydroxyvitamin D (25-OHD) in plasma (the most useful index of vitamin D status) was measured in six groups each of seven kittens given a purified diet (12 g calcium and 8 g phosphorus/kg, calculated metabolizable energy = 20 kJ/g) that contained either 0.0, 3.125, 6.25, 12.5, 18.75 or 25 microg of cholecalciferol/kg diet. All kittens received these diets from 9 to 22 wk of age, and the two groups given the 0.0 and 3.125 microg cholecalciferol/kg treatments continued to receive the diets until they were 34 wk old. Total and ionizable calcium and phosphorus in plasma were not affected by treatments. No adverse clinical changes were observed or found on radiographic examination of the kittens at 22 or 34 wk of age. Plasma concentration of 25-OHD was linearly related (r2 = 0.99, P < 0.001) to dietary intake of cholecalciferol. Plasma concentration of 25-OHD in kittens given the diet without added vitamin D was significantly less at 22 wk than at 9 wk, whereas kittens receiving the diet containing 3.125 microg cholecalciferol/kg had significantly higher 25-OHD concentrations at 22 and 34 wk than at 9 wk of age. Kittens given the 6.25 microg cholecalciferol/kg diet had plasma 25-OHD concentrations at 22 wk > 50 nmol/L which is considered replete for humans. An allowance of 6. 25 microg (250 IU) of cholecalciferol/kg diet is suggested to provide a margin of safety.  (+info)

Structure-function studies of new C-20 epimer pairs of vitamin D3 analogs. (4/960)

A growing number of calcitriol (1alpha,25-dihydroxyvitamin D3) analogs have become available in recent years. Many of these analogs exhibit lower calcemic effects than calcitriol and inhibit cell proliferation and enhance cell differentation more efficiently than calcitriol. We have compared structure-function relationships of a series of new C-20 epimer (20-epi) vitamin D3 analogs with their natural C-20 counterparts. In human MG-63 osteosarcoma cells, quantification of cellular osteocalcin mRNA levels by Northern blot analysis and osteocalcin biosynthesis by radioimmunoassay indicated that most studied analogs at a concentration of 10 nm induced osteocalcin gene expression more efficiently than the parent compound, calcitriol. Interestingly, when the biological responses were compared with the binding affinities of the analogs to in-vitro translated human vitamin D receptor and with their ability to protect the receptor against partial proteolytic digestion, significant correlations were not observed. Further, molecular modelling of the compounds by energy minimization did not reveal marked differences in the three-dimensional structures of the analogs. These results suggest that higher than normal ligand binding affinity or 'natural' conformation of the ligand-receptor complex are not necessarily required for the 'superagonist' transactivation activity. The mechanism of action of the efficient analogs may involve stabilization and/or differential binding of transcriptional coactivators or transcription intermediary factors to the hVDR during transactivation.  (+info)

1alpha,25-dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats. (5/960)

We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 as a major in vitro metabolite of 1alpha,25-dihydroxyvitamin D3, produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1alpha,25-dihydroxy-3-epi-vitamin D3 from the serum of rats treated with pharmacological doses of 1alpha,25-dihydroxyvitamin D3. 1alpha,25-dihydroxy-3-epi-vitamin D3 was identified through its co-migration with synthetic 1alpha,25-dihydroxy-3-epi-vitamin D3 on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1alpha,25-dihydroxy-3-epi-vitamin D3, other previously known metabolites, namely, 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxo-vitamin D3 and 1alpha,25-dihydroxyvitamin D3-26,23-lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1alpha,25-dihydroxy-3-epi-vitamin D3 is an in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats.  (+info)

Cloning of porcine 25-hydroxyvitamin D3 1alpha-hydroxylase and its regulation by cAMP in LLC-PK1 cells. (6/960)

The 25-hydroxyvitamin D3 1alpha-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 micromol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 micromol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1alpha,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1alpha,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.  (+info)

Vitamin D3 supplementation of beef steers increases longissimus tenderness. (7/960)

The objectives of these experiments were to determine 1) the effectiveness of supplemental vitamin D3 (VITD) on altering plasma and muscle calcium levels, 2) whether VITD supplementation improves Warner-Bratzler shear force (WBS) values of steaks from feedlot beef steers, and 3) the tenderness response curve of longissimus steaks from steers supplemented with VITD. In Exp. 1, 20 crossbred steers were assigned randomly to one of four treatment diets consisting of either 0, 2.5, 5.0, or 7.5 x 106 IU of VITD per day for 10 d. Blood samples were obtained daily during this supplementation period and 5 d thereafter (d 11 to 15). Between d 6 and 13, a linear increase (P < .01) in ionized plasma calcium concentrations was observed in steers supplemented with VITD. Compared to unsupplemented steers, serum calcium concentrations of the steers receiving 7.5 x 106 IU of VITD per day were increased 8 to 48%. In Exp. 2, longissimus samples from crossbred steers (n = 118) that were supplemented with either 0 or 5 x 106 IU of VITD per day for 7 d were obtained and aged for 7, 14, or 21 d. Following the initial 7-d postmortem aging period, VITD supplementation lowered (P < .01) WBS (.58 kg) and increased sensory tenderness rating (.6 units) compared to cuts originating from unsupplemented steers. In Exp. 3, 44 steers were supplemented with either 0 or 7.5 x 106 IU of VITD per day for 10 d immediately prior to slaughter. Results indicated that plasma and longissimus calcium concentration were higher (P < .05) for steers that received supplemental VITD. Compared with unsupplemented cuts, VITD supplementation improved WBS of cuts aged for either 7 or 14 d (P = .02 and P = .07, respectively). Sensory panelists rated samples from VITD supplemented steers as more tender than their unsupplemented counterparts. Activation of calpain proteases could be responsible for the observed tenderization due to the supplementation of VITD.  (+info)

A two-hit mechanism for vitamin D3-mediated transcriptional repression of the granulocyte-macrophage colony-stimulating factor gene: vitamin D receptor competes for DNA binding with NFAT1 and stabilizes c-Jun. (8/960)

We previously described a control element in the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhancer that is necessary and sufficient to mediate both transcriptional activation in response to T-cell stimuli and transcriptional repression by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through the vitamin D3 receptor (VDR). This DNA element is a composite site that is recognized by both Fos-Jun and NFAT1; it is directly bound by VDR in the absence of a retinoid X receptor as an apparent monomer, and it is bound in a unique tertiary conformation. We describe here the mechanism by which VDR elicits its transcriptional inhibitory effect. Firstly, VDR outcompetes NFAT1 for binding to the composite site. Overexpression of NFAT1 in vivo by transient transfection is able to relieve the 1,25(OH)2D3-dependent repression. Secondly, VDR stabilizes the binding of a Jun-Fos heterodimer to the adjacent AP-1 portion of the element. This appears to occur through a direct interaction between VDR and c-Jun, as demonstrated in vitro by direct glutathione S-transferase coprecipitation assays. In vivo, overexpression of c-Jun, but not c-Fos, leads to a rescue of the 1, 25(OH)2D3-mediated repression. Transfected FLAG-VDR bound to the NFAT1-AP-1 DNA binding element can be selectively precipitated from nuclear extracts that are made from cells treated with activating agents in the presence of 1,25(OH)2D3. VDR is not detected in the complex in the absence of the ligand. Thus, VDR acts selectively on the two components required for activation of this promoter/enhancer: it competes with NFAT1 for binding to the composite site, positioning itself adjacent to Jun-Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun's transactivation function. These two events mediated by VDR effectively block the NFAT1-AP-1 activation complex, resulting in an attenuation of activated GM-CSF transcription.  (+info)

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