Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol

Abstract: Human leukemia K562 cells can be induced to erythroid differentiation when treated with a variety of compounds, including hemin, cytosine arabinoside and 5-azacytidine. Following erythroid induction, K562 cells express at high level γ-globin and accumulate both Hb Portland and Hb Gower 1. In this paper we determined whether a combination treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids lead to full expression of differentiated functions. Cell growth kinetics studies, intracellular detection of hemoglobin by benzidine staining and hemoglobin analysis by cellulose acetate were performed. The results obtained show that (a) retinoic acid and retinol are not able to induce differentiaton of K562 cells and (b) cytosine arabinoside induces differentiation only when used at 100–300 nmol/l concentrations. In addition, our data demonstrate that erythroid differentiation of K562 occurs when 40 μmol/l of retinoic acid or retinol are added together with 75 nmol/l cytosine arabinoside.     

Distinct roles for IL-13 and IL-4 via IL-13 receptor alpha1 and the type II IL-4 receptor in asthma pathogenesis

IL-13 and IL-4 are central T helper 2 (Th2) cytokines in the immune system and potent activators of inflammatory responses and fibrosis during Th2 inflammation. Recent studies using Il13ra1(-/-) mice have demonstrated a critical role for IL-13 receptor (IL-13R) alpha1 in allergen-induced airway responses. However, these observations require further attention especially because IL-4 can induce similar lung pathology to IL-13, independent of IL-13, and is still present in the allergic lung. Thus, we hypothesized that IL-13Ralpha1 regulates IL-4-induced responses in the lung. To dissect the role of IL-13Ralpha1 and the type I and II IL-4Rs in experimental asthma, we examined lung pathology induced by allergen, IL-4, and IL-13 challenge in Il13ra1(-/-) mice. We report that IL-13Ralpha1 is essential for baseline IgE production, but Th2 and IgE responses to T cell-dependent antigens are IL-13Ralpha1-independent. Furthermore, we demonstrate that increased airway resistance, mucus, TGF-beta, and eotaxin(s) production, but not cellular infiltration, are critically dependent on IL-13Ralpha1. Surprisingly, our results identify a CCR3- and IL-13Ralpha1-independent pathway for lung eosinophilia. Global expression profiling of lungs from mice stimulated with allergen or IL-4 demonstrated that marker genes of alternatively activated macrophages are differentially regulated by the type I and type II IL-4R. Taken together, our data provide a comprehensive mechanistic analysis of the critical role by which IL-13Ralpha1 mediates allergic lung pathology and highlight unforeseen roles for the type II IL-4R.     

Variations in culture pH affect the cloning efficiency and differentiation of progenitor cells in ex vivo haemopoiesis

Haemopoietic cultures may experience pH variations of as much as 0.5 units depending on culture duration and cell density. Since pH is a potent modulator of cellular proliferation and differentiation, we examined its effects on the performance of both semisolid and liquid haemopoietic cultures. Culture pH was found to have substantial effects both on progenitor cloning efficiency (as measured in liquid cultures) and on progenitor cell differentiation (as measured in methylcellulose cultures). Liquid cultures were conducted with both peripheral blood (PB) mononuclear cells (MNCs) and cord blood (CB) MNCs using growth factor combinations that promote either erythroid expansion (IL-3/IL-6/SCF/Epo) or granulocyte/macrophage expansion (IL-3/IL-6/SCF/G-CSF/GM-CSF). Reduced pH was found to have either a positive or neutral effect on the expansion and cloning efficiency of progenitors in ex vivo liquid cultures. Cloning efficiencies of PB BFU-E in the erythroid combination were 9-fold higher at low pH (7.1) when compared to high pH (7.6). A small pH increase of 0.2 units over physiological values consistently produced significant reductions (42–85%) in cloning efficiencies for all cell types and cytokine combinations tested. Methylcellulose cultures conducted using CB MNC and PB MNC indicated that differentiation of CFU-GM into progeny was optimal between pH 7.2 and 7.4. The differentiation of erythroid progenitors (BFU-E) progressively increased as pH was increased from 6.95 (no colonies detected) to 7.4 (maximum colonies detected), to 7.6 (maximum haemoglobin content). Methylcellulose cultures using PB CD34⁺ cells exhibited similar patterns to the MNC cultures. We conclude that even small variations in pH substantially affected the performance of human haemopoietic cultures. The erythroid lineage was particularly sensitive, with its extent of differentiation increasing with increasing pH. PB progenitors are more sensitive to pH variations than CB progenitors.     

Pro-inflammatory (IL-1beta,IL-18) cytokines and IL-8 chemokine release by PBMC in response to Echinococcus multilocularis metacestode vesicles

In humans infected with Echinococcus multilocularis, larval metacestodes will develop, proliferate and progressively infiltrate the surrounding host tissues by exogenous budding of parasitic microvesicles or cell lines which detach from the original tumour and thus become transported through blood or lymph vessels into other organs. Cellular effector mechanisms constitute the most effective means to restrict parasite persistence and proliferation, and here we demonstrate that E. multilocularis vesicle antigens will induce pro-inflammatory, regulatory and chemokine release by PBMC from patients. The pro-inflammatory cytokines IL-1beta and IL-18 were reduced in echinococcosis patients, regulatory IL-10 was similar, but parasite vesicle-induced IL-8 was dominant and clearly elevated in patients. Such selective and opposite dynamics of inflammatory cytokines and chemokine release may prevent overwhelming and pathogenic inflammation, and constitute an appropriate response for attraction of effector cells into the periparasitic tissues with the capacity to limit E. multilocularis metacestode proliferation and dissemination.     

Ability of myeloma cells to secrete macrophage inflammatory protein (MIP)-1alpha and MIP-1beta correlates with lytic bone lesions in patients with multiple myeloma

Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta have been identified as candidates for multiple myeloma (MM)-derived bone-resorbing factors. To validate the clinical relevance of these observations, we investigated correlations between the ability of MM cells to secrete these chemokines and the extent of MM bone lesions as well as levels of biochemical bone markers in patients with MM. Patients with multiple bone lesions exhibited higher MIP-1alpha and MIP-1beta secretion from MM cells along with elevated urinary deoxypyridinoline (Dpd), without significant elevation of serum bone-specific alkaline phosphatase (BALP) or osteocalcin compared with those with minimal bone lesions. MIP-1alpha and MIP-1beta levels correlated positively with urinary Dpd and serum BALP but not with serum osteocalcin. These results provide further evidence for a causal role of MIP-1alpha and MIP-1beta in the development of lytic bone lesions, and suggest that MM cells suppress osteoblastic bone formation to cause an imbalance of bone turnover and development of destructive bone lesions.     

Characterization and localization of expression of an erythropoietin-induced gene, ERIC-1/TACC3, identified in erythroid precursor cells

Gene expression profiles during erythropoietin (Epo)-induced differentiation of erythroid progenitor cells derived from the Friend virus anaemia (FVA) and phenylhydrazine (PHZ) murine models have been examined using differential display polymerase chain reaction (PCR). Ten cDNA fragments upregulated by Epo were isolated. The ribonuclease protection assay confirmed differential expression between Epo-stimulated and Epo-deprived cells for one of these, provisionally named ERIC-1. Sequencing of the full-length cDNA predicted a protein of 558 amino acids, 17 amino acids longer than mTACC3, the third member of a novel family of proteins that contain a coiled-coil domain. The human homologue, cloned using rapid amplification of cDNA ends (RACE)-PCR, encodes a larger protein of 838 amino acids that is identical to hTACC3. In addition to erythroid precursor cells, ERIC-1/TACC3 is expressed at high levels in the testes, at moderate levels in the thymus and peripheral leucocytes, and at lower levels in the spleen and intestinal tissue. Immunohistochemical analysis using an antibody to a GST fusion product of the C-terminus of hERIC-1/TACC3 revealed that it is localized to Sertoli cells in the human testes. Confocal microscopy demonstrated hERIC-1/TACC3 protein concentrated in the perinuclear vesicles of dermal microvascular endothelial cells. Although ERIC-1/TACC3 is expressed in a wide range of tissues, its upregulation by Epo in erythroid progenitors implies that it has a role in terminal erythropoiesis.     

Bb1-3, a transgenic hybrid cell line with erythroid and megakaryocytic differentiation potential that expresses high levels of human γ-globin and human β-globin

We have characterized a murine hybrid cell line, Bb1-3, generated by the fusion of mouse primary erythroblasts with MEL cells. It proliferated in serum-free medium and displayed a low level of spontaneous erythroid and megakaryocyte differentiation. Terminal erythroid differentiation could be induced with HMBA and DMSO and was enhanced by serum. Treatment with phorbol esters resulted in a high proportion of megakaryocytes and the expression of megakaryocytic specific lineage markers. Bb1-3 cells contain a human β-globin transgene that was expressed at levels of 20–50% of the endogenous mouse globin genes. Initially, expression was largely limited to the β-globin gene but after adaptation to serum free growth, equal expression of both the human γ- and human β-globin genes was observed. This cell line provides further evidence that the differentiation potential of mouse erythroleukaemia cells is not restricted to the erythroid lineage and should be useful to study the mechanisms underlying both developmental globin gene regulation and the terminal differentiation of bipotential erythroid/megakaryocytic progenitor cells.     

Sensitization by 5-aza-2'-deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3

Most chromosomal abnormalities associated with breakage at 11q23 in acute leukaemia involve the MLL gene, and the presence of this breakage strongly predicts a poor clinical outcome. We assessed the possibility of differentiation-inducing therapy for acute leukaemias with chromosomal translocations involving 11q23. Among the cell lines with MLL translocations that we examined, KOCL48 and KOPN-1 cells were induced to differentiate into granulocytes by all-trans retinoic acid (ATRA) or into monocytes by 1alpha,25-dihydroxyvitamin D3 (VD3). These cells expressed p16 mRNA before treatment with 5-aza-2'-deoxycytidine (5-AZA), an inhibitor of DNA methylation. On the other hand, differentiation was not induced in SN-1, KOCL33, KOCL51 or KOCL44 cells by ATRA or VD3, and these cells did not express mRNA of this gene. However, these cells were effectively induced to differentiate by ATRA or VD3 in the presence of 5-AZA, and concomitantly exhibited p16 gene expression, suggesting an association between DNA demethylation and restoration of sensitivity to differentiation-inducing activity of ATRA or VD3 in leukaemia cells with MLL abnormalities. Based on these findings, combined treatment with ATRA or VD3 plus 5-AZA may be clinically useful in therapy for acute leukaemia with MLL abnormalities.     

High T-helper-1 cytokines but low T-helper-3 cytokines, inflammatory cytokines and chemokines in children with high risk of developing type 1 diabetes

BACKGROUND: Type 1 diabetes (T1D) is suggested to be of T-helper (Th)1-like origin. However, recent reports indicate a diminished interferon (IFN)-gamma secretion at the onset of the disease. We hypothesize that there is a discrepancy in subsets of Th-cells between children with a high risk of developing T1D, children newly diagnosed with T1D and healthy children. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from children at high risk for T1D (islet cells antibodies [ICA] >/= 20 IJDF-U), those newly diagnosed and healthy children carrying the HLA-risk gene DQB1*0302 or DQB1*0201 and DQA1*0501. Th1- (IFN-gamma, tumour necrosis factor [TNF]-beta, interleukin [IL]-2), Th2- (IL-4,-5,-13), Th3- (transforming growth factor [TGF-beta], IL-10) and inflammatory associated cytokines (TNF-alpha, IL-1alpha,-6) and chemokines (monocyte chemoattractant protein [MCP]-1,-2,-3, Monokine unregulated by IFN-gamma [MIG], Regulated on Activation, Normal T-cell Expressed and Secreted [RANTES], IL-7,-8,-15) were detected in cell-culture supernatants of PBMC, stimulated with glutamic acid decarboxylase 65 (GAD(65)) and phytohaemagglutinin (PHA), by protein micro array and enzyme linked immunospot (ELISPOT) technique. RESULTS: The Th1 cytokines IFN-gamma and TNF-beta, secreted both spontaneously and by GAD(65)- and mitogen stimulation, were seen to a higher extent in high-risk children than in children newly diagnosed with T1D. In contrast, TNF-alpha and IL-6, classified as inflammatory cytokines, the chemokines RANTES, MCP-1 and IL-7 as well as the Th3 cytokines TGF-beta and IL-10 were elevated in T1D children compared to high-risk children. CONCLUSION: High Th-1 cytokines were observed in children with high risk of developing TID, whereas in children newly diagnosed with T1D Th3 cytokines, inflammatory cytokines and chemokines were increased. Thus, an inverse relation between Th1-like cells and markers of inflammation was shown between children with high risk and those newly diagnosed with T1D.     

Serum interleukin (IL)-1, IL-2, sIL-2Ra, IL-6 and thrombopoietin levels in patients with chronic myeloproliferative diseases

A number of growth factors are involved in clonal haematopoietic expansion and their clinical significance in patients with chronic myeloproliferative diseases requires further evaluation. Using enzyme-linked immunosorbent assays, we analysed serum levels of interleukin (IL)-1a, IL-1b, IL-2, IL-6, the soluble IL-2 receptor alpha (sIL-2Ra), and thrombopoietin (TPO), in 25 individuals with myelofibrosis with myeloid metaplasia (MMM), 40 with essential thrombocythaemia (ET), eight with polycythaemia vera (PV), 10 patients with chronic myeloid leukaemia (CML) and 27 normal controls. These were correlated with clinicopathological characteristics including overall survival, and histopathological bone marrow features, including angiogenesis. The serum derived from patients with MMM, ET, PV and CML contained significantly higher IL-2 and sIL-2Ra than healthy subjects, while IL-6 levels were higher only in MMM and CML than controls. IL-2, sIL-2Ra and IL-6 levels were raised during the transformation phase of CML, during progression of MMM to AML, and ET and PV to myelofibrosis (P < 0.001). There was a positive correlation between IL-2, sIL-2Ra, IL-6 and angiogenesis in bone marrow samples. Cytokines may be useful markers for predicting clinical evolution, reflecting increased angiogenesis. This requires further evaluation to guide diagnostic and therapeutic options.     

TNF-α, IL-8, soluble ICAM-1, and neutrophils in sputum of cystic fibrosis patients

The cytokines tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and intercellular adhesion molecule-1 (ICAM-1) have important roles in regulating neutrophil migration and the inflammatory response. To determine whether the concentration of these cytokines and soluble ICAM-1 (sICAM-1) in sputum was increased in patients with cystic fibrosis during acute exacerbations, we conducted (1) a cross-sectional study of 40 patients, 22 who were clinically well and 18 with acute pulmonary exacerbations; and (2) an 11 months longitudinal study of 16 patients. Significant differences in clinical scores, pulmonary function, and sputum neutrophil density were found between the acutely ill and the well group. There was a strong linear relationship (P < 0.0005) between TNF-α and IL-8 concentrations in sputum, but no association between clinical status and cytokine concentrations. The concentration of sICAM-1 was lower in acutely ill compared with well patients in the cross-sectional study. Recovery of exogenous IL-8 added to sputum was complete, while recovery of TNF-α averaged 70%. Recovery of exogenous sICAM-1 was only 43%, and the recoveries were lower in sputum samples from acutely ill patients than those from stable patients (P = 0.018). These data indicate that in cystic fibrosis patients, sputum concentrations of TNF-α and IL-8 are not increased during acute exacerbations of pulmonary inflammation. Pediatr Pulmonol. 1996; 21:11–19 . © 1996 Wiley-Liss, Inc.     

阿尔茨海默病患者血清与脑脊液中多种细胞因子的研究

目的　探讨阿尔茨海默病(Alzheimer’sdisease ,AD)病因及发病机制。方法　采用双抗体夹心酶联免疫吸附试验分别对5 7例患者分为AD组和VaD组进行血清和脑脊液(CSF)肿瘤坏死因子-α(TNF- α)、白细胞介素- 1β(IL- 1β)及白细胞介素- 8(I-L 8)水平检测。对照组2 8例均无神经系统、免疫系统等疾病。结果　(1)AD组CSF中TNF- α水平明显高于VaD组和对照组,差异有显著性意义(P <0 .0 5 ,P <0 .0 1) ;(2 )AD组和VaD组CSF中IL- 1β水平明显高于对照组(P <0 .0 5 ) ,AD组血清中IL -1β水平明显高于VaD组和对照组,差异有显著性意义(P <0 .0 1) ;(3)AD组和VaD组血清和CSF中IL 8均明显高于对照组,差异显著(P <0 .0 5 ,P <0 .0 1) ;(4)AD组患者CSF中的TNF -α、IL -1β及IL -8水平之间呈正相关(r =0 .0 6 ,P <0 .0 5 ) ;(5 )血清或CSF中的TNF- α、IL- 1β及IL -8检测水平与简易智力状态检查表(MMSE)得分之间均无相关性。结论　细胞因子可能参与了AD的病理生理过程。     

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells

Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen‐activated protein kinases (MAPKs) [extracellular signal‐regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3‐kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12‐myristate 13‐acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA‐treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbα expression, DNA endoreduplication, and formation of platelet‐like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down‐regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.     

Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

BACKGROUND AND OBJECTIVE: The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. DESIGN AND METHODS: Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. RESULTS: An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. INTERPRETATION AND CONCLUSIONS: The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.     

Prostaglandin E1 and F2 alpha stimulate differentiation and proliferation, respectively, of clonal osteoblastic MC3T3-E1 cells by different second messengers in vitro

The effect of several prostaglandins (PGs) on osteoblastic cells was investigated using clone MC3T3-E1 under serum-free conditions. PGA1, A2, B1, and B2 had little effect on intracellular cAMP, alkaline phosphatase (ALP) activity, and DNA synthesis in the cells. At 4-2000 ng/ml, PGE1 among PG analogs tested had a dose-dependent stimulatory effect on ALP activity in the cells, and this effect was amplified by isobutyl methylxanthine. Also, PGE1 strongly augmented the amount of intracellular cAMP over the same concentration range. However, PGE1 had little effect on ornithine decarboxylase activity and DNA synthesis, and at high doses it rather depressed DNA synthesis. Furthermore, PGE1 did not affect the intracellular cGMP level. The effect of PGE1 on the cells closely mimics that of forskolin, suggesting that the PG stimulates the differentiation of the osteoblastic cells predominantly via the stimulation of adenylate cyclase. In contrast with PGE1, PGF2 alpha strongly increased ornithine decarboxylase activity and DNA synthesis in the cells in a dose-related fashion at low concentrations (4-100 ng/ml), at which concentrations it had little effect on the intracellular cAMP or cGMP level and depressed ALP activity. Moreover, PGF2 alpha depressed the stimulatory effect of PGE1 on ALP activity but did not affect the elevation of cAMP level by PGE1. The accumulation of inositol phosphates was greatly increased by PGF2 alpha in the concentration range effective in stimulating DNA synthesis, but was increased little by PGE1, suggesting that PGF2 alpha is a potent stimulator of phosphatidyl inositol turnover in the cells. In addition, A23187, a Ca ionophore, alone did not influence the DNA synthesis, but the effects of tetradecanoyl phorbol acetate, a direct activator of protein kinase C, were very similar to those of PGF2 alpha. Moreover, the stimulation of DNA synthesis or the inhibition of ALP activity by PGF2 alpha was partially counteracted by H-7, a strong inhibitor of protein kinase C. These results suggest that PGF2 alpha stimulates the proliferation of osteoblastic cells predominantly through the phosphatidyl inositol turnover system following in part the activation of protein kinase C. Our data presented here indicate that PGE1 and PGF2 alpha are closely involved in the differentiation and proliferation, respectively, of osteoblasts in vitro and that their action may be mediated by second messengers which differ from each other.     

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1alpha,25-dihydroxyvitamin D3

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK₁ kidney cells. To induce 24-hydroxylase expression, 1,25(OH)₂D₃ (10⁻⁷ M) and dexamethasone were added simultaneously to the medium of LLC-PK₁ cells, and 24 h before dexamethasone treatment, 1,25(OH)₂D₃ was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells. Maximal stimulation was observed with 10⁻⁶ M dexamethasone in both cell lines. The addition of 10⁻⁶ M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10⁻⁶ M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)₂D₃-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)₂D₃-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)₂D₃ enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.     

Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/SCID mice) transplanted intravenously with 10⁷ human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34⁺ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18–20 weeks. ³H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4–6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo .