Specific-heat critical behavior of the structural random-field system Dy(AsxV1-x)O4

Vitamin D3 derivative 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) exerts various biological effects in cells that possess vitamin D3 receptor (VDR), including enhancement of cell differentiation and inhibition of cell proliferation. These activities of 1,25(OH)2D3 might be responsible for its anti-neoplastic effects, as shown in various experimental systems. The aim of this study was to compare the anti-angiogenic activity of 1,25(OH)2D3, retinoids, and interleukin-12 (IL-12) in an experimental tumor cell-induced angiogenesis assay in mice. Tumor cell-induced angiogenesis assay was performed in x-ray immunosuppressed BALB/c mice by intradermal injections of human tumor cell lines of different origin. The injections resulted within 3 d in a local formation of new blood vessels, and the intensity of angiogenesis correlated with the number of injected cells. Systemic treatment of the mouse recipients with 1,25(OH)2D3 significantly decreased angiogenesis, comparable to the effect of retinoids (all-trans retinoic acid [RA], 9-cis RA and 13-cis RA) and of IL-12. In vitro preincubation of the cells with all compounds (except IL-12) led to the inhibition of their angiogenic capability in vivo. Moreover, combination of 1,25(OH)2D3 and retinoids resulted in a synergistic inhibition of angiogenesis. The results strongly suggest that anti-angiogenic compounds with relatively low toxicity (e.g., 1,25(OH)2D3, retinoids, and IL-12) and their combinations could be beneficial in the treatment of some angiogenesis-associated malignancies.     

Apoptosis induction of POS canine osteosarcoma cells by vitamin D and retinoids

Vitamin D3: 1-alpha, 25(OH)2D3 (calcitriol), 22-oxa-1,25(OH)2D3 (OCT), cholecalciferol (vitamin D3), and retinoids: all-trans retinoic acid (ATRA) and 9-cis retinoic acid, induced morphological changes in POS canine osteosarcoma cells into elongated, spindle or fibroblast like-shaped cells, and apoptotic like cell death characterized by cell shrinkage, condensation and margination of the nucleus for all drugs at 10(-6)M-10(-9)M after 72 to 120 hr culture. Apoptosis as shown by DNA laddering was induced at 48 hr by all drugs at 10(-6)M, 10(-7)M at 96 hr, 10(-8)M and 10(-9)M at 120 hr respectively. These vitamins are suggested to adjunct antineoplastic agents in canine osteosarcoma therapy by induction of apoptosis.     

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.     

Combinational effects of vitamin D3 and retinoic acid (all trans and 9 cis) on proliferation, differentiation, and programmed cell death in two small cell lung carcinoma cell lines

The effects of a combination of vitamin D3 [1,25(OH)2D3] and retinoic acid (RA) on proliferation, differentiation, and apoptosis of the human small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H209 were evaluated. Cell proliferation was inhibited by 1,25(OH)2D3 and RA alone. The combination of 1,25(OH)2D3 and the cis form of retinoic acid resulted in an additive decrease in cell proliferation and the induction of apoptosis in various concentrations. Moreover, 3H-thymidine incorporation was inhibited and the number of viable cells was decreased. The characteristics of the apoptotic cells were examined and confirmed by morphologic analysis, light and electron microscopy, and fluorescence detection. It was concluded that 1,25(OH)2D3 and RA exert additive effects on the inhibition of proliferation and the induction of apoptosis in both the NCI-H82 and the NCI-H209 SCLC cell lines. This finding has important implications for the use of retinoids and 1,25(OH)2D3 in cancer prevention and in the therapy of small cell lung carcinoma.     

Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1

The secosteroid hormone, 1,25-dihydroxyvitamin D [1,25(OH)2D], plays a crucial role in normal bone growth, calcium metabolism, and tissue differentiation. The key step in the biosynthesis of 1,25(OH)2D is its 1 alpha-hydroxylation from 25-hydroxyvitamin D (25-OHD) in the kidney. Because its expression in the kidney is very low, we cloned and sequenced cDNA for 25-OHD-1 alpha-hydroxylase (P450c1 alpha) from human keratinocytes, in which 1 alpha-hydroxylase activity and mRNA expression can be induced to be much greater. P450c1 alpha mRNA was expressed at much lower levels in human kidney, brain, and testis. Mammalian cells transfected with the cloned P450c1 alpha cDNA exhibit robust 1 alpha-hydroxylase activity. The identity of the 1,25(OH)2D3 product synthesized in transfected cells was confirmed by HPLC and gas chromatography-mass spectrometry. The gene encoding P450c1 alpha was localized to chromosome 12, where the 1 alpha-hydroxylase deficiency syndrome, vitamin D-dependent rickets type 1 (VDDR-1), has been localized. Primary cultures of human adult and neonatal keratinocytes exhibit abundant 1 alpha-hydroxylase activity, whereas those from a patient with VDDR-1 lacked detectable activity. Keratinocyte P450c1 alpha cDNA from the patient with VDDR-1 contained deletion/frameshift mutations either at codon 211 or at codon 231, indicating that the patient was a compound heterozygote for two null mutations. These findings establish the molecular genetic basis of VDDR-1, establish a novel means for its study in keratinocytes, and provide the sequence of the key enzyme in the biological activation of vitamin D.     

Arthroscopic reattachment of the meniscus

Basic fibroblast growth factor (bFGF) has been detected in bone cells and stimulates osteoblast proliferation; however, its role in the regulation of bone metabolism remains speculative. We demonstrated that the human osteocalcin promoter is activated by bFGF when transfected into rat osteoblastic (ROS 17/2.8) cells. This effect is concentration dependent, with a twofold induction at 10 ng/ml detected after 20 h. The bFGF response is independent of both the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and retinoic acid activation of the osteocalcin promoter. To identify the promoter sequences through which bFGF exerts its effect, we tested a series of promoter deletion constructs for their response to bFGF. Deletion of the upstream region between -673 and -588 bp results in a significant loss of induction. Gel-shift analysis demonstrates that proteins present in ROS 17/2.8 nuclear extracts bind specifically to these sequences. This region alone was unable to confer the bFGF response on a minimal osteocalcin or an heterologous promoter. However, sequences between -678 and -476 bp, which also includes the vitamin D response element (VDRE), were able to confer bFGF inducibility on both a minimal osteocalcin and a heterologous promoter. These data suggest that induction of the human osteocalcin promoter by bFGF requires the interaction of more than one sequence element.     

1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.     

The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.     

Distribution and metabolism of F6-1,25(OH)2 vitamin D3 and 1,25(OH)2 vitamin D3 in the bones of rats dosed with tritium-labeled compounds

26,26,26,27,27,27-Hexafluo-1,25(OH)2 vitamin D3, the hexafluorinated analog of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biologic activity in the kidneys and small intestine appears to be related to F6-1,25(OH)2 vitamin D3 metabolism to ST-232, 26,26,26,27,27,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3, a bioactive 23S-hydroxylated form that is resistant to further metabolism. Since F6-1,25(OH)2 vitamin D3 is considered to prevent osteoporotic decrease in bone mass by suppressing bone turnover, we here compared the distribution and metabolism of [1 beta-3H]F6-1,25(OH)2 vitamin D3 and [1 beta-3H]1,25(OH)2 vitamin D3 in bones of rats by autoradiography and radio-HPLC. In the dosed groups, radioactivity was detected locally in the metaphysis, the modeling site in bones. As compared with the [1 beta-3H]1,25(OH)2 vitamin D3 case, [1 beta-3H]F6-1,25(OH)2 vitamin D3 was significantly retained in this site, and moreover, it mainly persisted as unchanged compound and ST-232. These findings indicate that the reason for the higher potency of F6-1,25(OH)2 vitamin D3 than 1,25(OH)2 vitamin D3 in bones are linked with increased distribution and reduced metabolism.     

Detection of a human chromosomal translocation t(8;9) in a baby with multiple malformations using two-color fluorescence in situ hybridization

In human fibroblast cultures TPA increased IL-6 and IL-8 production. This was reduced by vitamin D3 metabolites and analogs. The two analogs employed: 1,25 (OH)2-22 (E)-dehydro-24-monohomo vitamin D3 (Compound A) and 1,25 (OH)2 -22 (E)-dehydro-24 dihomo-vitamin D3 (Compound B) may be useful in the therapy of pathologic proliferative disorders including psoriasis, particularly since they are less toxic and have less effect on calcium metabolism than vitamin D3.     

1,25-dihydroxyvitamin D3 stimulates the assembly of adherens junctions in keratinocytes: involvement of protein kinase C

Signaling via intercellular junctions plays an important role in the regulation of growth and differentiation of epithelial cells. Loss of cell-cell contacts has been implicated in carcinogenesis, tumor progression, and metastasis. Here, we investigated whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was able to stimulate the assembly of adherens junctions and/or desmosomes in cultured human keratinocytes. After 4-day incubation, 1,25-(OH)2D3 caused assembly of adherens junctions, but not desmosomes. The adherens junctions were identified upon known ultrastructural criteria and evidence of the translocation of specific junctional proteins (E-cadherin, P-cadherin, alpha-catenin, and vinculin) to the cell-cell borders. The presence of alpha-catenin and vinculin at cell-cell borders indicated that the adherens junctions were functional. This was further supported by showing that anti E-cadherin antibody inhibited the 1,25-(OH)2D3-induced keratinocyte stratification. A relation between protein kinase C and adherens junction regulation was noticed. 1,25-(OH)2D3-dependent formation of junctions was blocked by the inhibitors of protein kinase C, bisindolylmaleimide and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), and treatment of keratinocytes with 1,25-(OH)2D3 caused a rapid activation of protein kinase C and its translocation to the membranes. Formation of intercellular contacts may be an important mechanism of 1,25-(OH)2D3 action in hyperproliferative and neoplastic diseases.     

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.