B. Surendra Baliga

p38 MAP kinase activation mediates γ-globin gene induction in erythroid progenitors

Abstract Objective Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on γ-globin gene activity was studied. Method Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on γ-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on γ-globin mRNA synthesis was analyzed. Results NB and TSA activated p38 MAPK and increased γ-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and γ-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased γ-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. Conclusion This study supports a novel role for p38 MAPK in γ-globin regulation in human erythroid progenitors.

In vivo and in vitro iron deficiency reduces protein kinase C activity and translocation in murine splenic and purified T cells

We investigated the effects of iron deficiency anemia, iron repletion, and iron chelation by deferoxamine on protein kinase C (PKC) activity, an enzyme that plays a crucial role on T lymphocyte proliferation. The study involved 23 control (C), 18 pairfed (PF), and 24 iron deficient (ID) mice or ID mice that were repleted for 3 (n = 14), 7 (n = 17), or 14 (n = 14) days. The low iron (0.09 mmol iron/kg) and iron‐supplemented (0.9 mmol iron/kg) diets were fed to mice for 53 days. Mean hemoglobin, hematocrit, and liver iron stores of ID mice were one third of those of C mice. Lymphocyte proliferation was reduced (P < 0.05) in spleen and purified T cells in ID but not PF mice. In concanavalin A, phytohemagglutinin, and anti‐CD3 antibody‐treated and untreated cells that were incubated in serum‐free and serum‐containing medium, PKC activity was significantly (P < 0.05) reduced in ID but not PF mice and returned to normal before correction of anemia. In mitogen‐treated cells, while the ratios of membrane‐bound to cytosol activity increased nearly seven‐fold (from 0.4–0.63 in resting cells to 1.43–7.23) in spleen cells from C, PF, and repleted mice and 11‐fold in T cells (P < 0.005), they remained below 1 in ID mice suggesting reduced translocation. In vitro iron chelation by deferoxamine for 120 min prior to cell activation reduced (P < 0.05) PKC activity by 46–60% in C and PF and 28–53% in ID mice. The data suggest that: 1) it is iron‐deficiency but not anemia or differences in the proportion of immunocompetent T cells that reduced PKC activity in cells from ID mice; 2) reduced PKC translocation may play an important role on altered lymphocyte proliferation and associated functions in iron‐deficient individuals. J. Cell. Biochem. 74:468–478, 1999.

BFU‐E colony growth in response to hydroxyurea: Correlation between in vitro and in vivo fetal hemoglobin induction

Patients with sickle‐cell anemia treated with hydroxyurea may have significant reduction in frequency and severity of pain episodes. However, previous clinical trials show a variable response to hydroxyurea. Criteria which can be used to select patients who are likely to respond to hydroxyurea treatment would be useful. Our laboratory has previously demonstrated an inverse linear relationship between the total number of burst‐forming unit‐erythroid (BFU‐E) colonies and fetal hemoglobin levels in sickle‐cell patients treated with hydroxyurea. In the present report, an in vitro cell culture system was established to evaluate the effects of hydroxyurea on BFU‐E colony growth and induction of fetal hemoglobin production. Five Hb SS patients who were not previously treated with hydroxyurea and three Hb SS patients who failed to respond to hydroxyurea treatment were included in the study. The results show that the number of BFU‐E colonies is decreased from 153.7 to 7.2 per 3 × 105 mononuclear cells, whereas fetal hemoglobin levels were increased from 5.1 to 19.4% in the presence of hydroxyurea in vitro in cultured erythroid progenitors, which were derived from 5 patients before treatment. The number of BFU‐E colonies decreased from 153.7 to 2.0 per 3 × 105 mononuclear cells in the in vitro cultures obtained from serial peripheral blood samples over a 9‐ to 20‐week period of oral hydroxyurea therapy. A simultaneous rise in fetal hemoglobin level from 10.2 to 28.6% in the peripheral blood over the same period of hydroxyurea therapy was also observed. Our results demonstrate that the increase in fetal hemoglobin levels in cells treated with hydroxyurea in vitro is comparable to the rise of fetal hemoglobin production following hydroxyurea therapy in these patients. On the contrary, these findings were not observed in three previously non‐responsive sickle‐cell patients. These results suggest that the changes in number of BFU‐E colonies and fetal hemoglobin levels after in vitro exposure to hydroxyurea may be a useful approach to select sickle‐cell patients who will respond to hydroxyurea therapy. Am. J. Hematol. 56:252–258, 1997.

Mechanism for fetal hemoglobin induction by hydroxyurea in sickle cell erythroid progenitors

Hydroxyurea (HU) is a widely used cytotoxic agent that is known to induce fetal hemoglobin (HbF) production and is presently used to ameliorate the severity of pain episodes in patients with sickle cell anemia (HbSS). Previously we have shown that HU inhibits growth of burst forming unit–erythroid (BFU‐E) colonies in a dose‐dependent manner, while fetal hemoglobin levels were increased. In the present report, we extended our analysis demonstrating the number of S phase cells is significantly higher for HbSS patients that respond to HU therapy. Studies were completed in vitro using erythroid progenitors derived from umbilical cord samples or peripheral blood from patients with HbS–hereditary persistence of fetal hemoglobin (HbS‐HPFH) or HbSS disease. The effect of HU on (a) S phase erythroid progenitors, (b) BFU‐E colony growth, (c) HbF levels in BFU‐E colonies, and (d) total cellular RNA synthesis was analyzed in vitro for the three groups. The level of S phase erythroid progenitors was similar for all three groups and BFU‐E colony growth was inhibited 92–94% for all samples in a dose‐dependent manner. The HbF levels were increased in BFU‐E colonies from HbSS patients (control, 4.0% ± 1.15% vs. +HU, 22.67% ± 2.03%) whereas HbF levels were decreased in BFU‐E colonies derived from umbilical cord samples (control, 80% ± 9.07% vs. +HU, 35.7% ± 4.81%) or HbS‐HPFH patients (control, 49.67% ± 3.84% vs. +HU, 23.3% ± 0.88%). Total RNA synthesis measured by 3H‐uridine incorporation increased with increasing concentrations of HU; however, actinomycin D inhibited HU‐induced RNA synthesis. These results suggest that HU can inhibit an active globin gene without preference and that newly synthesized RNA is under transcriptional control mechanisms. Am. J. Hematol. 65:227–233, 2000.

Reduced thymocyte proliferation but not increased apoptosis as a possible cause of thymus atrophy in iron-deficient mice.

Iron deficiency induces thymus atrophy in laboratory animals and very likely in humans by unknown mechanisms. The atrophy is associated with impaired cell-mediated immunity. In this study, we tested the hypothesis that thymus atrophy is a result of increased apoptosis and reduced thymocyte proliferation. Thymocytes were obtained from twenty-seven control, twenty-seven pairfed, twenty-seven iron-deficient (ID) mice; twelve and fourteen ID mice that received the control diet (0.9 mmol/kg versus 0.09 mmol/kg for the ID diet) for 1 d (repletion, R1) and 3 d (R3), respectively. Cell cycle analysis and apoptosis were studied by flow cytometry using propidium iodide staining and terminal deoxyuridine nick end labeling of DNA breaks assay respectively. When mice were killed, haemoglobin, haematocrit, and liver iron stores of ID, R1, and R3 mice were 25-40 % of those of control and pairfed mice Absolute and relative thymus weights and thymocyte numbers were 19 to 68 % lower in ID, R1, and R3 than in control and pairfed groups We found no significant difference among groups in the percentage of cells undergoing apoptosis. A higher percentage of thymocytes from ID and R1 mice than those of control, pairfed, and R3 mice were in the resting phase of the normal cell cycle Conversely, a lower percentage of thymocytes from ID and R1 mice than those from control, pairfed, and R3 mice were in the DNA synthesis phase and late phase of DNA synthesis and onset of mitosis (G2-M) Indicators of iron status positively correlated (r 0.3 to 0.56) with the percentage of thymocytes in the G2-M phase Results suggest that reduced cell proliferation but not increased apoptosis is the cause of thymus atrophy associated with iron deficiency.

Effects of growth and differentiation inducing factors on protein kinase-C of cultured intestinal crypt cells

To assess the role of protein kinase-C (PK-C) in the growth and differentiation of small intestinal enterocytes, IEC-6 cells (a cell line derived from the crypts of rat small intestine) were incubated with factors known to induce growth (insulin, epidermal growth factor, gastrin, somatostatin and transferrin) or differentiation (transforming growth factor-beta, retinoic acid and phorbol 12-myristate 13-acetate (PMA)). Cell proliferation (3H-thymidine incorporation) and PK-C activity (Ca++/phospholipid dependent) were measured. Among growth promoting factors only epidermal growth factor, insulin and transferrin were associated with increased 3H-thymidine incorporation, and none of these agents induced PK-C activation as measured by its translocation from cytosol to membrane fraction. Of the differentiation inducing factors, only PMA translocated PK-C from cytosol to membrane. PMA also inhibited 3H-thymidine incorporation in a dose dependent manner. These results suggest that growth and proliferation of enterocytes occur independent of PK-C signal transduction.

Effect of polymyxin-B on T-lymphocyte protein synthesis

The role of protein kinase-C (PK-C) protein phosphorylation on the mitogen triggered responses of T-lymphocytes was examined by observing the effect of polymyxin-B (an inhibitor of PK-C) on mitogen induced protein and DNA synthesis. Polymyxin-B inhibited 3H-thymidine incorporation by PHA activated T-lymphocytes over a range of PHA concentrations. 3H-leucine incorporation by PHA activated T-lymphocytes was inhibited by polymyxin-B in a dose dependent manner. A partially purified PK-C fraction from polymyxin-B treated PHA activated T-lymphocytes demonstrated less than 25% of the phosphorylating activity of untreated lymphocytes. These results suggest that protein synthesis during the T-lymphocyte activation process is dependent on PK-C activity.

Differences in iron requirements by concanavalin A-treated and anti- CD3-treated murine splenic lymphocytes

Fe availability is critical for optimal lymphocyte proliferation; however, the minimum required levels are unknown. Such information is valuable when assessing in vitro immune responses in Fe-deficient subjects, because serum (Fe) added to the culture medium may replete lymphocytes. To address this issue, splenic lymphocytes obtained from seventeen 3-month-old C57BL/6 mice were incubated without and with 1 mg/l concanavalin A or 50 microg/l anti-CD3 antibody in media that contained between 0.113 and 9.74 micromol Fe/l. Fe was provided by either fetal calf serum (FCS, 0-100 ml/l), newborn calf serum (NBCS, 0-100 ml/l), or NBCS (10 ml/l) plus ferric ammonium citrate. As expected, the rate of DNA synthesis increased with Fe levels (P<0.01). Maximum DNA synthesis was obtained with 2.26 micromol Fe/l (50 ml FCS/l) for concanavalin A and 0.895 micromol/l (20 ml FCS/l) for anti-CD3-treated cells. In serum-free media (0.113 micromol Fe/l), the proliferative responses to concanavalin A were below the background, while they rose 5.5-fold in anti-CD3-treated cells (P<0.05). In apotransferrin-supplemented media (0.13 micromol Fe/l), the proliferative responses to concanavalin A and anti-CD3 antibody were 18.6 and 71 %, respectively, of that obtained with 4.66 micromol Fe/l (100 ml FCS/l). Interleukin 2 secretion also followed the same trend as lymphocyte proliferation. Since differences between both mitogens persisted after FCS was substituted with NBCS, we can rule out an effect on ribonucleotide reductase activity, or by other serum growth factors. We speculate an Fe effect at an early step of T-cell activation. Data suggest that the minimum Fe concentration required for lymphocyte proliferation varies with the mitogen.

Synthesis and processing of RNA by isolated human placental nuclei

Conditions for isolating intact and active nuclei from human term pacenta and for studying their transcription products are described. The isolated nuclei can synthesize cell-free RNA for a prolonged period at 29 degree C in a medium containing 100 mM KCl and 5 mM MgCl2. Actinomycin D inhibited 92 per cent of RNA synthesis, whereas approximately 60 per cent of RNA synthesis was sensitive to alpha-amanitin. When nuclei were incubated at 29 degrees C for 1 h, about 27 per cent of the newly synthesized RNA was released into the medium outside the nucleus. Analysis of this released material by affinity chromatography on an oligo(dT)-cellulose column revealed that 2.4 per cent of the total released RNA was adsorbed at high salt concentration. Most of this fraction was eluted with a low-salt buffer at 45 degrees C and the remainder by 50 per cent formamide, conditions that are necessary for elution of poly(A)-containing mRNP particles from oligo(dT)-cellulose. These results show that placental nuclei incubated in vitro synthesize poly(A)-containing RNA, which is released as a protein-bound complex. This procedure allows exploration of changes in mRNA release during placental development.

970 EFFECT OF DIETARY IRON ON CELLULAR IMMUNE FUNCTION

The effect of inadequate dietary iron uptake on cellular immune responses was studied in weanling female mice C57BL/6, which were fed either control (C), iron deficient (DEF) or pairfed (PF) C diets. The DEF mice showed a significant decrease in the delayed cutaneous hypersensitivity reaction to dinitroflurobenzene (DNFB) as measured by the inflammatory skin response (p<.001) and 125I-dUR incorporation into sensitized ears (p<.001). When a single dose of imferon was given to DEF mice prior to recall, the 125I-dUR incorporation was restored to greater than normal levels (p<.001), a response which was DNA, RNA, protein synthesis dependent (Table 1).When the cytotoxic capacity of T-lymphocytes or peritoneal cells to kill 51Cr labelled P815 target tumor cells were compared, the cells from the DEF group had a killing capacity which was 50% of the C. The above data strongly supports the contention that iron deficiency significantly depresses the in vivo and in vitro CMI response.* M ± SEM.