NORMAL AND MALIGNANT BREAST TISSUE IS A TARGET ORGAN FOR 1, 25-(OH)2 VITAMIN D3

Malignant and benign human breast tumours as well as rabbit breast tissue were examined for specific receptors for 1,25-dihydroxyvitamin D3 using competitive binding studies and sucrose density gradient analysis. Classical high affinity, low capacity receptors for 1,25-dihydroxyvitamin D3 were found in breast and node tissue in seven of ten patients with breast cancer and in all three patients with benign neoplasms. An inflammatory breast mass showed no binding. Similar receptors were found in breast tissue from pregnant and lactating rabbits. Taken with other recent data, these results suggest that 1,25-dihydroxyvitamin D3 may activate calcium transport in the malignant as well as in the normal lactating breast.     

Myeloid progenitors from the bone marrow of patients with vitamin D resistant rickets (type II) fail to respond to 1,25(OH)2D3

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to enhance the growth of human granulocyte/macrophage haemopoietic progenitors in vitro and to induce these cells to differentiate along the monocyte/macrophage pathway. In order to evaluate the relationship between specific receptors for 1,25(OH)2D3 and the role of 1,25(OH2D3 in the regulation of haemopoietic cell differentiation, we examined the effect of haemopoietic cell differentiation, we examined the effect of 1,25(OH)2D3 on the in vitro growth and differentiation patterns of marrow myeloid progenitor cells from two patients with 1,25(OH)2D3 resistant rickets, resulting from defective receptors to vitamin D. A significant rise in the frequency of myeloid colonies in control marrow cell cultures was induced by 2 X 10(-9) to 2 X 10(-7)M 1,25(OH)2D3. This rise reached a plateau at 2 X 10(-9)_2 X 10(-8) M 1,25(OH)2D3, resulting in a maximal 54 +/- 9% increase in colony numbers. In contrast, no stimulatory effect could be detected when 1,25(OH)2D3 was added to cultured marrow cells from the patients with 1,25(OH)2D3 resistance. Analysis of colony composition revealed that 2 X 10(-8) and 2 X 10(-7) M, 1,25(OH)2D3 induced a 50 +/- 26% increase in the frequency of colonies composed only of monocytes/macrophages in control, but not in the patients' marrow cell cultures. The effect of 2 X 10(-8) and 2 X 10(-7) M 1,25(OH)2D3 on progenitor cell differentiation towards monocytes/macrophages was also observed in marrow cell suspension cultures. Whereas 1,25(OH)2D3 induced a 81-136% increase in the frequency of monocytes in control marrow cells, no effect could be detected on the generation of mature monocytes in marrow cells of the 1,25(OH)2D3 resistant patients. Our results show that marrow granulocyte/macrophage progenitor cells from patients with 1,25(OH)2D3 resistance fail to respond to 1,25(OH)2D3. We thus demonstrate that the effect of 1,25(OH)2D3 on the proliferation and differentiation of haemopoietic progenitor cells is mediated through its binding to specific cytoplasmic receptors.     

Gene expression analysis of 1,25(OH)2D3-dependent differentiation of HL-60 cells: a cDNA array study

The alterations in gene expression associated with 1,25(OH)2D3-induced differentiation of HL-60 cells were studied in order to identify potential targets for further investigation of the genetic basis of acute myeloid leukaemia. Atlas human haematology filters, including 406 genes (Clontech), were used to study gene expression in response to 1,25(OH)2D3 (concentration, 5 x 10-8 mol/l) for 24 and 72 h. Compared with untreated cells, expression differences were found in 43 genes. Downregulated genes at both time-points were: IL2RA, CMYC, NPM, DEK, AF4, FLI1, htlf, MNDA, BCR, IKAROS, BPI and NFAT4. Upregulated genes at both time-points were IL1B, CD14 and MCL1. CD55, CD58, IRF2, CREB1, ATF4, RAC1, TIAR, KIAA0053, BAT2, BTK, RCK, EV12B and EDN were downregulated at 24 h, while SPI1, MKK3, BTG1 and IL8 were upregulated. At 72 h the upregulated genes were IL1RA, IL2RG, CXCR4, SCYA1, SCYA3, SCYA4, SCYA5, SCYA22, ANX2, CD83 and UPAR. cDNA array results were confirmed on randomly selected genes using quantitative real-time polymerase chain reaction for three upregulated (CXCR4, IL1B and CD14) and three downregulated (DEK, AF4 and FLI1) genes. Gene expression analysis after differentiation induction may provide a tool to study the roles of DEK, AF4 and FLI1 in cell proliferation and differentiation. To demonstrate the genes that initiate differentiation, sequential gene expression analysis has to be performed during the first 24 h of the differentiation process.     

A common Vδ2—Dδ2—Dδ3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) alpha, beta and gamma chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, delta, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the delta chain gene locus, using probes of the variable, joining, and constant regions of the delta chain gene. The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR delta genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR delta gene rearrangements were detected. An unusual TCR delta rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR delta rearrangements. In contrast to the utilization ov V delta 1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of J delta segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive V delta 2-D delta 2-D delta 3 recombination in this precursor B ALL. We conclude that the TCR delta chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.     

Effect of vitamin D2 and vitamin D3 on the serum concentrations of 1,25(OH)2D2, and 1,25(OH)2D3 in normal subjects

Serum concentrations of vitamin D2 and vitamin D3 metabolites were measured in 19 normal subjects before and during treatment with either vitamin D2 or vitamin D3, 4000 IU per day for 8 weeks. Vitamin D2 treatment increased the serum concentration of 1,25(OH)2D2, but a corresponding decrease in 1,25(OH)2D3 resulted in an unchanged serum concentration of total 1,25(OH)2D. During treatment with vitamin D3, the serum concentration of 1,25(OH)2D metabolites was unchanged. We conclude that the production of 1,25(OH)2D is tightly regulated and that 1 alpha-hydroxylase does not discriminate between D2 and D3 metabolites in normal subjects.     

α4β1 integrin-mediated adhesion of CD34+ cells from patients with chronic myeloid leukaemia: influence of IL-3

Interactions between integrins on haemopoietic progenitor cells and their stromal ligands have an important role in the control of haemopoiesis. Growth factors can modulate these interactions (so-called 'inside-out' signalling) resulting in changes in ligand binding activity. We have studied alpha4beta1 integrin-mediated adhesion to the H120 fragment of fibronectin (which contains the strongest alpha4beta1 binding site) in CD34+ cells from patients with chronic myeloid leukaemia (CML) and have determined the effect of IL-3 on the level of adhesion. Compared to normal CD34+ cells isolated from cord blood and peripheral blood progenitor harvests (mean of 61.4 +/- 14.9% of cells attached) the CML CD34+ cells showed reduced levels of adhesion (mean of 41.9 +/- 14.7%, P < 0.05). The effect of 10 ng/ml of IL-3 resulting in reduced adhesion of normal CD34+ cells at 30 min was absent in 6/7 patients with CML. Abnormalities of adhesion to fibronectin may thus be related to IL-3 pathways affected by BCR-ABL. These findings will have implications for understanding the dysregulation of growth and adhesion in CML.     

Intermittent intravenous followed by intermittent oral 1α(OH)D3 treatment of secondary hyperparathyroidism in uraemia

Brandi L, Daugaard H, Egsmose C, Tvedegaard E, Kjærulff Nielsen P, Olgaard K (Medical Department P, Division of Nephrology, Rigshospitalet, University of Copenhagen, Denmark). Intermittent intravenous followed by intermittent oral 1α(OH)D₃ treatment of secondary hyperparathyroidism in uraemia. J Intern Med 1996; 239: 353–60. Objectives . To examine whether intermittent oral 1α(OH)D₃ treatment of patients on haemodialysis with secondary hyperparathyroidism (HPT) was able to maintain the marked suppression of PTH, which previously had been induced by an intermittent intravenous administration of 1α(OH)D₃. Simultaneously, the effect of the different routes of administration of 1α(OH)D₃ on the circulating levels of N-and C-terminal PTH fragments was measured. Design . An open study of patients on chronic haemodialysis. Setting . Renal division, Rigshospitalet, Copenhagen, Denmark. Subjects . A total of 26 patients started and five patients completed the total protocol. Interventions . The treatment protocol was divided into three parts: (i) 1α(OH)D₃ administered intravenously for >300 days; then (ii) 1α(OH)D₃ administered orally for 100 days, followed by (iii) 1α(OH)D₃ administered intravenously again for another 100 days. 1α(OH)D₃ was given three times a week at the end of each dialysis. Main outcome measures . Intact PTH, N- and C-terminal PTH. Results . Intact PTH levels were significantly (P<0.0001) suppressed by 90.4±3.3% after 56 days of intermittent intravenous 1α(OH)D₃ treatment. This degree of suppression remained stable during the following period of oral treatment and did not change further when intravenous treatment was reinstituted. The circulating levels of intact PTH and N- and C-terminal iPTH were not influenced by the administered route of 1α(OH)D₃. Conclusions . Intravenous 1α(OH)D₃ treatment of the secondary HPT in dialysis patients can safely be changed to oral treatment at the time when optimal suppression of PTH has been achieved.     

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10⁻⁷ M, 1,25(OH)₂D₃ for 24 h resulted in a 20–30% decrease in PKI mRNA. 1,25(OH)₂D₃ treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)₂D₃ directly and tissue-specifically downregulates PKI mRNA in the chick kidney.     

The cyclin-dependent kinase inhibitors p18INK4c and p19INK4d are highly expressed in CD34+ progenitor and acute myeloid leukaemic cells but not in normal differentiated myeloid cells

Cyclin-dependent kinase inhibitors (CKIs) are important for the differentiation of cells in various tissues. In acute myeloid leukaemia (AML) the cells accumulate at particular stages of myeloid maturation. We therefore analysed the expression pattern of different CKIs in fresh samples of AML patients and compared it with that in CD34+ progenitor and normal differentiated myeloid cells. Competitive RT-PCR and Western analysis revealed a significantly higher expression of p18INK4c and p19INK4d in leukaemic and CD34+ progenitor cells than in granulocytes and monocytes. A different pattern was seen for p27Kip1 and p57Kip2 expression being low in leukaemic cells but high in normal immature and differentiated cells. No marked differences were found in p15INK4b and p21Cip1 mRNA expression between leukaemic and CD34+ progenitor or mature myeloid cells. Our findings therefore indicate that high expression of p18INK4c and p19INK4d in haemopoietic progenitor and leukaemic blast cells may contribute to the premature differentiation block seen in AML.     

Regulation of cell growth, c-myc mRNA, and 1,25-(OH)2 vitamin D3 receptor in C3H/10T1/2 mouse embryo fibroblasts by calcipotriol and 1,25-(OH)2 vitamin D3.

Calcipotriol is a synthetic 1,25-(OH)2D3 analogue with high affinity for the 1,25-(OH)2D3 receptor, but with a lower affinity than 1,25-(OH)2D3 for vitamin D binding protein in serum. The inhibitory action of calcipotriol and 1,25-(OH)2D3 on proliferation of C3H/10T1/2 mouse embryo fibroblasts was examined in the non-transformed cell line Cl 8 and in the two transformed, tumorigenic cell lines Cl 16 and TPA 482. Upon exposure to 10 nmol/l calcipotriol or 1,25-(OH)2D3, the proliferation of Cl 8 cell line was almost completely suppressed, whereas both hormones had no effect on the cell lines Cl 16 and TPA 482. Calcipotriol was at least as effective as 1,25-(OH)2D3 in inducing up-regulation of the 1,25-(OH)2D3 receptor. Displacement studies showed no difference between calcipotriol and 1,25-(OH)2D3 in the affinity for the receptor present in Cl 8 or Cl 16 cell extracts. Furthermore, the inhibition of cell growth in Cl 8 cells by calcipotriol was not accompanied by any consistent change in the steady-state expression of c-myc mRNA. In conclusion, calcipotriol had potent growth inhibitory effect on the non-transformed cell line similar to 1,25-(OH)2D3. In the transformed cell lines, calcipotriol did not inhibit proliferation despite potent up-regulation of the 1,25-(OH)2D3 receptor.     

1,25-二羟基维生素D3和维生素K2联合诱导HL-6O细胞分化及凋亡的研究

目的研究1,25-二羟基维生素D3[1,25(OH)2D3,简称D3]与维生素K2(VK2)联合应用对HL-60细胞分化及凋亡的影响.方法通过四唑氮蓝(MTT)比色,细胞形态,流式细胞仪(FCM)测定细胞周期、凋亡率及CD14的表达,观察D3、VK2对HL-60细胞的影响.结果D3与VK2都能抑制HL-60细胞增殖,并且联合使用抑制作用显著.10-8mol/L D3与10 μmol/L VK2联合处理HL-60细胞72 h后CD14的表达率为63.15%,且G0/G1期细胞显著增多,与单独一种比较差异有统计学意义(P＜0.05).10 μmol/L、20 μmol/L VK2作用于HL-60细胞72 h凋亡率分别为11.31%、20.36%,与对照组比较差异有统计学意义(P＜0.01);而10 μmol/LVK2与10-8 mol/L D3联用72 h细胞的凋亡率为5.41%,与对照组比较差异无统计学意义(P＞0.05).结论D3与VK2联合使用可以使D3诱导分化作用加强,而VK2诱导凋亡作用受到抑制.     

1,25(OH)2vitamin D3 stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells

Skeletal muscle wasting is an important public health problem associated with aging, chronic disease, cancer, kidney dialysis, and HIV/AIDS. 1,25-Dihydroxyvitamin D (1,25-D3), the active form of vitamin D, is widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance and for its calcemic effects on target organs, such as intestine, kidney, and parathyroid glands. Emerging evidence has shown that vitamin D administration improves muscle performance and reduces falls in vitamin D-deficient older adults. However, little is known of the underlying mechanism or the role 1,25-D3 plays in promoting myogenic differentiation at the cellular and/or molecular level. In this study, we examined the effect of 1,25-D3 on myoblast cell proliferation, progression, and differentiation into myotubes. C(2)C(12) myoblasts were treated with 1,25-D3 or placebo for 1, 3, 4, 7, and 10 d. Vitamin D receptor expression was analyzed by quantitative RT-PCR, Western blottings and immunofluorescence. Expression of muscle lineage, pro- and antimyogenic, and proliferation markers was assessed by immunocytochemistry, PCR arrays, quantitative RT-PCR, and Western blottings. Addition of 1,25-D3 to C(2)C(12) myoblasts 1) increased expression and nuclear translocation of the vitamin D receptor, 2) decreased cell proliferation, 3) decreased IGF-I expression, and 4) promoted myogenic differentiation by increasing IGF-II and follistatin expression and decreasing the expression of myostatin, the only known negative regulator of muscle mass, without changing growth differentiation factor 11 expression. This study identifies key vitamin D-related molecular pathways for muscle regulation and supports the rationale for vitamin D intervention studies in select muscle disorder conditions.     

Haemoglobin Inkster (α285 aspartic acid → valineβ2) Coexisting with β-Thalassaemia in a Caucasian Family

S ummary . Haemoglobin Inkster, a new α-chain variant, was discovered in a family which also had the gene for β-thalassaemia. The amino acid abnormality was in αTp-9 which contains 29 amino-acid residues. Structural studies were facilitated by cleavage of the abnormal α-chains with cyanogen bromide followed by tryptic digestion. The substitution was shown to be valine for aspartic acid at position 85 in the α-chain. Affected individuals had no haematological abnormalities. Individuals with both β-thalassaemia and Hb Inkster had slightly lower percentages of Hb Inkster than those found in persons heterozygous for the Hb Inkster gene alone. 'Interaction' between thalassaemia and variant haemoglobin genes involving different haemoglobin loci has been reported in another family with β-thalassaemia and an α-chain haemoglobin mutant, as well as in the converse situation of coexisting β-thalassemia and a β-chain haemoglobin mutant. This decrease in the mutant haemoglobin percentage differs from the more common 'interaction' of thalassaemia and mutant haemoglobin genes involving the same haemoglobin locus, in which the mutant haemoglobin percentage is increased. The mechanism for the 'interaction' is unknown, but the presence of an unusually low percentage of a haemoglobin variant should warrant investigation for coexisting thalassaemia involving a different haemoglobin locus.     

The Role of α1 Protease Inhibitor (α1 Antitrypsin) in the Regulation of Immunologic and Inflammatory Reactions

Summary: Twenty-six different alleles have been identified for α₁ protease inhibitor (α₁ antitrypsin), each designated by a letter of the alphabet. In any individual two alleles co-dominantly determine the characteristics of α₁ protease inhibitor (Pi), including mobility on electrophoresis, serum concentration and acute phase response. Recent evidence has linked some mildly deficient phenotypes of Pi with a variety of chronic immunologic and inflammatory disorders, such as rheumatoid arthritis, juvenile chronic arthritis, systemic lupus erythematosus, ankylosing spondylitis, uveitis, asthma and fibrosing alveolitis, in addition to the well recognised association of severe deficiency with emphysema and chronic liver disease. This disease susceptibility in phenotypes associated with reduced serum levels may be due to alteration in lymphocyte responses, complement activation and leukocyte migration. Pi can also influence the autolytic effects of leukocytic enzymes on tissues and may inhibit some aspects of coagulation and fibrinolysis. Therefore patients with deficient Pi phenotypes are likely to have exaggerated immunologic and inflammatory responses.     

Adriamycin stimulates proliferation of human lymphoblastic leukaemic cells via a mechanism of hydrogen peroxide (H2O2) production

It is becoming clear that adriamycin cytotoxicity may be mediated by semiquinone-free radicals derived from the drug itself and reactive oxygen species (ROS). Recent evidence supports the concept that low concentrations of ROS are able to stimulate cell proliferation, and, based on the observation that subtoxic concentrations of adriamycin can also induce cell proliferation, we hypothesize that low concentrations of adriamycin stimulate cell proliferation by a ROS generation mechanism. We have employed spin-trapping and electron spin resonance (ESR) spectroscopy to investigate the nature of the adriamycin-generated ROS. The spin trap 3,5-dibromo-4-nitrosobenzenesulphonate (DBNBS), which is oxidized in the presence of H₂O₂ and peroxidase enzymes, was used to produce a characteristic three-line spectrum, and it was found that an identical spectrum was produced by human lymphoblastic leukaemic cells (CCRF-CEM cells) after exposure to adriamycin. We tested our hypothesis further by exposing CCRF-CEM cells to subtoxic concentrations of adriamycin (10⁻⁸, 10⁻⁹ and 10⁻¹⁰  M ) and low concentrations of H₂O₂ (10⁻⁸, 10⁻⁹ and 10⁻¹⁰  M ) and subsequently monitored cell proliferation. We found that low concentrations of both adriamycin and H₂O₂ significantly stimulate CCRF-CEM cell proliferation. We therefore conclude that subtoxic concentrations of adriamycin are likely to induce cell proliferation via an H₂O₂ mediated mechanism.