Concentrations Of Hydroxyurea Research Articles

In vivo pharmaco-proteomic analysis of hydroxyurea induced changes in the sickle red blood cell membrane proteome.

Hydroxyurea (HU) is an effective drug for the treatment of sickle cell disease (SCD). The main clinical benefit of HU is thought to derive from its capacity to increase fetal hemoglobin (HbF) production. However, other effects leading to clinical benefit, such as improved blood rheology, have been suggested. In order to understand HU-induced changes at the proteomic level, we profiled sickle RBC membranes from of HU-treated and untreated patients. Our previous in vitro profiling studies on sickle RBC membranes identified a significant increase in predominantly anti-oxidant enzymes, protein repair and degradation components and a few RBC cytoskeletal proteins. In the present study, using 2D-DIGE (Two-Dimensional Difference In-Gel Electrophoresis) and tandem mass spectrometry, we detected 32 different proteins that significantly changed in abundance in the HU treatment group. The proteins that significantly increased in abundance were mostly membrane skeletal components involved in the regulation of RBC shape and flexibility, and those showing a significant decrease were components of the protein repair and degradation machinery. RBC palmitoylated membrane protein 55 (p55) is significantly increased in abundance at low (in vitro) and high (in vivo) concentrations of HU. Palmitoylated p55 may be an important target of HU-dependent regulation of the sickle RBC membrane, consistent with our earlier in vitro studies.

A non‐apoptotic function of caspase‐3 in pharmacologically‐induced differentiation of K562 cells

Several anticancer drugs with diverse chemical structures can induce differentiation of cancer cells. This study was undertaken to explore the potential contribution of caspase-3 to pharmacologically-induced differentiation of K562 cells. We assessed differentiation by measuring the expression of glycophorin A and haemoglobin synthesis in K562 cells treated with low concentrations of doxorubicin, hydroxyurea, cytosine arabinoside, cisplatin and haemin. Caspase-3 activation, mitochondrial membrane potential dissipation and viability were assessed by FACS. GATA-1-binding activity was evaluated by EMSA. Treatment of K562 cells with low concentrations of the tested drugs activated caspase-3 but did not trigger detectable apoptosis. Instead, elevated levels of haemoglobin-positive and glycophorin A/caspase-3-double-positive cells were observed, suggesting involvement of caspase-3 in drug-induced differentiation. Inhibition of caspase-3 activity significantly reduced the ability of K562 cells to execute the differentiation programme. Mitochondrial membrane potential dissipation was observed, indicating involvement of the mitochondrial pathway. Binding activity of GATA-1, transcription factor responsible for differentiation and cell survival, was not diminished by increased caspase-3 activity during drug-stimulated differentiation. Our results could explain how anticancer drugs, with diverse structures and modes of action, can stimulate erythroid differentiation in leukaemic cells with appropriate genetic backgrounds. Our findings imply that some similarities exist between pharmacologically-induced differentiation of erythroleukaemic cells and normal erythropoiesis, both involving caspase-3 activation at high levels of anti-apoptotic protein Bcl-X(L) and chaperone protein Hsp70 (heat shock protein 70). Therefore, the functions of caspase-3, unrelated to cell death, can be extended to pharmacologically-induced differentiation of some cancer cells.

Cyanide, Peroxide and Nitric Oxide Formation in Solutions of Hydroxyurea Causes Cellular Toxicity and May Contribute to Its Therapeutic Potency

Hydroxyurea (HU) is a potent remedy against a variety of ailments and an efficient inhibitor of DNA synthesis, yet its pharmacology is unclear. HU acts in Escherichia coli by the same mechanism as it does in eukaryotes, via inhibition of ribonucleotide reductase. When examining a controversy about concentrations of HU that prevent thymineless death in E. coli, we found instability in HU solutions that avoided prior detection due to its peculiar nature. In contrast to freshly dissolved HU, which did not affect respiration and was bacteriostatic, 1-day-old HU solutions inhibited respiration and were immediately bactericidal. Respiration was inhibited by two gases, hydrogen cyanide (HCN) and nitric oxide (NO), whose appearance we detected in “aged” HU stocks by gas chromatography–mass spectrometry; however, neither gas was bactericidal. While determining the cause of toxicity, we found that HU damages DNA directly. We also demonstrated accumulation of peroxides in HU solutions by enzymatic assays, which explains the toxicity, as both NO and HCN are known to kill bacteria when combined with hydrogen peroxide. Remarkably, we found that bactericidal effects of NO+H2O2 and HCN+H2O2 mixtures were further synergistic. Accumulation of decomposition products in solutions of HU may explain the broad therapeutic effects of this drug.

Pharmaco-proteomic study of hydroxyurea-induced modifications in the sickle red blood cell membrane proteome.

Hydroxyurea (HU) is an effective oral drug for the management of homozygous sickle cell anemia (SS) in part because it increases fetal hemoglobin (HbF) levels within sickle red blood cells (RBCs) and thus reduces sickling. However, results from the Multicenter Study of HU suggested that clinical symptoms often improved before a significant increase in HbF levels occurred. This indicated that HU may be acting through the modification of additional cellular mechanisms that are yet to be identified. Hence, in this study, we focused on the analysis of the sickle RBC membrane proteome +/- HU treatment. 2D-DIGE (Two Dimensional Difference In-Gel Electrophoresis) technology and tandem mass spectrometry has been used to determine quantitative differences between sickle cell membrane proteins in the presence and absence of a clinically relevant concentration of HU. In vitro protein profiling of 13 sickle RBC membrane samples +/- 50 muM HU identified 10 statistically significant protein spots. Of these, the most remarkable class of proteins to show a statistically significant increase was the anti-oxidant enzymes-catalase, thioredoxin peroxidase and biliver-din reductase and the chaperonin containing TCP1 complex assisting in the folding of RBC cytoskeletal proteins. Interestingly, catalase immunoblots showed an increase in the acidic forms of the enzyme within sickle RBC membranes on incubation with 50 muM HU. We further identified this modification in catalase to be phosphorylation and demonstrated that HU exposed SS RBC membranes showed a 2-fold increase in tyrosine phosphorylation of catalase as compared to counterparts not exposed to HU. These results present an attractive model for HU-induced post-translational modification and potential activation of catalase in mature sickle RBCs. These findings also identify protein targets of HU other than fetal hemoglobin and enhance the understanding of the drug mechanism.

Distinct Phenotypes of Hydroxyurea Absorption among Children with Sickle Cell Anemia

Hydroxyurea therapy is now used commonly for both adults and children with sickle cell anemia (SCA), and has both laboratory and clinical efficacy. At doses ranging from 10–35 mg/kg/day, biologically important laboratory responses to hydroxyurea include induction of fetal hemoglobin (HbF), increased hemoglobin concentration, decreased circulating white blood cell and reticulocyte counts, and reduction of hemolysis (lower LDH). Treatment with hydroxyurea at the maximum tolerated dose (MTD) provides optimal laboratory responses in children with SCA. Despite the proven efficacy of this therapeutic option, hydroxyurea has a variable effect among patients with SCA that is not well understood. Patients have variability in their HbF and LDH responses, but also in the toxicity profile, which typically manifests as myelosuppression and limits the MTD. To understand better the variation in hydroxyurea responses and toxicities, we performed first-dose pharmacokinetic (PK) studies on a cohort of children with SCA who were initiating hydroxyurea therapy for clinical indications. As part of the prospective IRB-approved research protocol Hydroxyurea Study of Long-term Effects (HUSTLE, ClinTrials.gov NCT00305175), 40 pediatric patients received a single 20 mg/kg oral dose of hydroxyurea (range 18.0–21.6 mg/kg) and had timed venous blood collection at 0, 30, 60, 120, 240, and 480 minutes. Serum was assayed for hydroxyurea concentration using a colorimetric chemical assay. Estimates of PK parameters for hydroxyurea were derived from individual concentration-time data sets using model independent methods. Maximum plasma concentration (Cmax) and the time of maximum concentration (Tmax) were observed; area under the concentration-time curve (AUC) was calculated using the final quantifiable sample and also by extrapolation to infinity. Additional PK parameters included terminal half-life (t1/2), apparent volume of distribution (V/F), and apparent oral clearance (CL/F). For all 40 patients, the apparent oral clearance (CL/F) of hydroxyurea was 0.252 ± 0.080 L/hr/kg, which is slightly increased compared to previously published values for adults. Hydroxyurea was rapidly absorbed after oral administration with a median time to peak concentration of 0.55 hours (range, 0.467–2.2 hours). Interindividual PK variability of hydroxyurea was substantial; the coefficient of variation was 26.0% for t1/2, 30.3% for Cmax, 46.7% for CL/F, and 61.6% for V/F. Variability in apparent oral clearance was related primarily to patient weight; linear regression indicated R-squared value of 0.533 indicating that over half of the observed interindividual variability could be accounted by weight alone. There was no significant contribution from hepatic or renal function, including glomerular filtration rate. Two distinct PK profiles for hydroxyurea were observed when patients were grouped on the basis of time to peak concentration: rapid and slow absorption phenotypes. For 25 children, the peak hydroxyurea concentration was observed at the first sampling time point, suggesting very rapid absorption; using a linear one-compartment model the predicted median time to peak concentration was 18 minutes. In these patients with the rapid absorption profile, the median Cmax was 74% higher than for the slow absorption (P<0.001) and the median dose-normalized AUC was 33% higher (p=0.037). These novel findings suggest that the rate of absorption of hydroxyurea is an important determinant of systemic exposure and possibly also of pharmacodynamic effects in children with SCA. Studies are ongoing to determine the effect of PK interindividual variation on HbF and LDH responses, as well as hydroxyurea MTD.

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.

The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity

The DNA-replication inhibitors aphidicolin (APC) and hydroxyurea (HU) were tested for their ability to induce effects on DNA in the in vitro alkaline comet assay with V79 cells. APC concentrations up to 15 μM and HU concentrations up to 500 μM did not significantly increase the extent of DNA migration after treatment during 4 h. Treatment for 18 h, however, led to inconsistently significant increase in DNA migration. These increases in DNA migration were accompanied by severe cell-cycle disturbances, cytotoxic effects (reduced population doubling and reduced mitotic index) and increased frequencies of cells with chromosome aberrations. The results indicate that substances with such secondary effects on DNA (in contrast to agents that directly damage DNA) only induce effects in the comet assay after prolonged exposure, together with cytotoxic effects. We conclude that slight inhibition of DNA replication and cell-cycle delay per se do not cause significant effects in the in vitro comet assay under standard test conditions. Furthermore, the in vitro comet assay seems to be less sensitive towards this type of secondary DNA effects than the in vitro chromosome aberration test.

An improved method for the cell cycle synchronization of Vicia faba root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of 3H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.

Hst3 Is Regulated by Mec1-dependent Proteolysis and Controls the S Phase Checkpoint and Sister Chromatid Cohesion by Deacetylating Histone H3 at Lysine 56

The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.

P53 Mediates Senescence-Like Arrest Induced by Chronic Replicational Stress

Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development.

Functional and Physical Interactions between Yeast 14-3-3 Proteins, Acetyltransferases, and Deacetylases in Response to DNA Replication Perturbations

The highly conserved 14-3-3 proteins participate in many biological processes in different eukaryotes. The BMH1 and BMH2 genes encode the two functionally redundant Saccharomyces cerevisiae 14-3-3 isoforms. In this work we provide evidence that defective 14-3-3 functions not only impair the ability of yeast cells to sustain DNA replication in the presence of sublethal concentrations of methyl methanesulfonate (MMS) or hydroxyurea (HU) but also cause S-phase checkpoint hyperactivation. Inactivation of the catalytic subunit of the histone acetyltransferase NuA4 or of its interactor Yng2, besides leading to S-phase defects and persistent checkpoint activation in the presence of genotoxic agents, is lethal for bmh mutants. Conversely, the lack of the histone deacetylase subunit Rpd3 or Sin3 partially suppresses the hypersensitivity to HU of bmh mutants and restores their ability to complete DNA replication in the presence of MMS or HU. These data strongly suggest that reduced acetyltransferase functionality might account for the S-phase defects of bmh mutants in the presence of genotoxic agents. Consistent with a role of 14-3-3 proteins in acetyltransferase and deacetylase regulation, we find that acetylation of H3 and H4 histone tails is reduced in temperature-sensitive bmh mutants shifted to the restrictive temperature. Moreover, Bmh proteins physically interact, directly or indirectly, with the Esa1 acetyltransferase throughout the cell cycle and with the Rpd3 deacetylase specifically during unperturbed S phase and after HU treatment. Taken together, our results highlight a novel role for 14-3-3 proteins in the regulation of histone acetyltransferase and deacetylase functions in the response to replicative stress.

Replication-deficient Adenovirus Induces Host Topoisomerase I Activity: Implications for Adenovirus-mediated Gene Expression

Replication-deficient adenoviruses are useful vectors for the transfer of therapeutic transgenes to malignant and non-malignant tissues. Yet their clinical application is limited by the potential toxicity of viral infection and the transient nature of transgene expression. Although transgene expression from adenovirus vectors is initially higher than expression of transgenes transduced by other viral or non-viral vectors, it is often insufficient to generate a significant therapeutic effect. We addressed this issue by searching for DNA-targeted viral-induced host responses potentially restricting transgene expression. Nuclear protein extracts from livers of rats systemically infected with replication-deficient adenovirus exhibited enhanced topoisomerase I activity compared with extracts from uninfected animals. Consequently, the inhibition of topoisomerase I by the anti-cancer drug topotecan greatly enhanced transgene expression in adenovirus-infected hepatic cells, colon cancer and prostate cancer cell cultures, mouse liver, human ex vivo tumor specimens, and mouse tumor in vivo. The enhancement could not be ascribed to non-specific genotoxic stress, cell death, or cell-cycle perturbation. These findings are significant for gene therapy as they reveal novel aspects of the host anti-adenovirus response and set the stage for the development of a rational molecular-pharmacological approach to increase the effectiveness, and safety, of adenovirus-mediated cancer therapeutics.

Plasma and Urine Hydroxyurea Levels Might be Useful in the Management of Adult Sickle Cell Disease

Hydroxyurea (HU) is useful for treating sickle cell anemia because of its ability to reduce some of the severe clinical events such as painful crises and acute chest syndrome. It may also reduce the need for blood transfusions and frequent hospitalizations and reduce mortality. Nevertheless, no consistent recommendations regarding its therapeutic schedule are defined. Our aim was to improve and validate a high performance liquid chromatography (HPLC) technique to measure HU and to study HU levels in serum and urine of sickle cell anemia patients and relate this to treatment efficacy and compliance. Thirty-seven patients received 1,128 ± 333 mg of HU per day (8.0 to 28.0 mg/kg/day). Plasma and/or urine were sampled and HU was measured using an HPLC method coupled with UV detection. We validated a specific, sensitive assay with good reproducibility and linearity, and showed a positive relationship between plasma HU concentrations and time elapsed between oral HU intake and sampling. We observed plasma HU concentrations were positively correlated with change in mean corpuscular volume (MCV) before and during the treatment. No correlation was obtained between HU concentration and Hb F level.

Hydroxyurea Induced Expression of gamma Globin and Erythrocyte Membrane Proteins in Cultured Erythroid Progenitor Cells.

BACKGROUND: Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell anemia and beta-thalassemia. The clinical efficacy of HU is due primarily to increases in fetal hemoglobin (HbF) levels. HU increases the %HbF and the %F cells. The HU reactive mechanism(s) in erythroid cells, however, have not been clearly defined. Patients receiving HU therapy develop subpopulations of macrocytic erythrocytes. Our previous studies demonstrate that sickle cell patients treated with low dose HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. The frequency of cells expressing greater levels of (AE1) per cell was increased in each serial blood sample. We propose that part of the HU reactive mechanism will include the upmodulation of non-gamma globin erythroid proteins that contribute to the macrocytic structures. As part of our investigation of the development of RBCs expressing increased band 3 protein per cell, we examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors.METHODS: HU induced protein synthesis was investigated as a function of HU concentration. Erythroid progenitors were cultured in serum free semisolid media containing different concentrations of HU [0–40 micromolar]. BFU-E were scored and harvested at day15 in culture, then assayed. BFU-E derived cells were first labeled with monoclonal anti-band 3 antibody. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry. In separate studies BFU-E derived cells were fixed, permeabilized, labeled with anti-spectrin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody. The labeled cells were assayed by confocal microscopy. For the next set of experiments, BFU-E cultured in increasing concentrations of HU were assayed for the presence of band 3 protein and gamma globin. BFU-E cells were fixed, permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody; then assayed by flow cytometry.RESULTS: Results show that the frequency of cells positive for band 3 protein [AE1] was increased at increasing concentrations of HU as compared to controls. The band-3 upmodulation appears to start to plateau at 12.5 micromolar HU. Band 3 protein derived from BFUE was confirmed by Western blot analysis. The frequency of cells positive for both band 3 and spectrin was similarly increased in cells cultured in the presence of increasing concentrations of HU. In separate studies, cells were assayed for the expression of both gamma globin and AE1. Results show a 2–3-fold increase in the % band 3 [AE1] plus gamma globin positive cells in HU [5–40 micromolar] treated cells as compared to untreated controls.CONCLUSIONS: These results demonstrate that HU induces the expression of band 3 and gamma globin in cultured erythroid progenitors. These findings suggest that part of the mechanism of HU action in erythroid cells involves the induction of erythroid structural proteins concordant with the induction of gamma globin.

SUMO-binding Motifs Mediate the Rad60-dependent Response to Replicative Stress and Self-association

In fission yeast, the replication checkpoint is enforced by the kinase Cds1 (human Chk2), which regulates both cell cycle progression and DNA repair factors to ensure that the genome is faithfully duplicated prior to mitosis. Cds1 contains a forkhead-associated domain that mediates its interaction with phosphorylated residues in target proteins. One target of Cds1 is the essential nuclear protein Rad60, which contains the unique structural feature of tandem SUMO homology domains at its C terminus. Hypomorphic mutants of Rad60 cause profound defects in DNA repair and replication stress tolerance. To explore the physiological significance of the Cds1-Rad60 interaction, we have examined the phosphorylation of Rad60 by Cds1 in vitro and the in vivo phosphorylation of Rad60 in response to replication blocks. We find that the N terminus but not the SUMO-like domain of Rad60 is phosphorylated in both conditions. Three important Rad60 phosphorylation sites were identified: Thr(72), Ser(32), and Ser(34). Rad60 Thr(72) mediates the Cds1-Rad60 interaction and is required for the Cds1-dependent phosphorylation of Rad60 in response to replication arrest. Phosphorylation of Rad60 Ser(32) and Ser(34) in a putative SUMO-binding motif is critical for the survival of replication stress. In addition, mutation of Rad60 Ser(32) and Ser(34) to alanine is lethal in cells deleted for the RecQ DNA helicase Rqh1. Finally, we find that Rad60 self-associates via its C-terminal SUMO-like domain and putative SUMO-binding motifs.

Modifications of H3 and H4 during Chromatin Replication, Nucleosome Assembly, and Histone Exchange

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the "histone code" associated with chromatin replication and dynamic histone exchange in human cells.

Rat liver-mediated metabolism of hydroxyurea to nitric oxide

Hydroxyurea is an approved treatment for sickle cell disease. Oxidation of hydroxyurea results in the formation of nitric oxide (NO), which also has drawn considerable interest as a sickle cell disease therapy. Although patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of crude rat liver homogenate to convert hydroxyurea to nitrite/nitrate, evidence for NO production. Nitrite/nitrate form at therapeutic concentrations of hydroxyurea in a clinically relevant time frame. Electron paramagnetic resonance (EPR) studies show the formation of iron nitrosyl complexes during this incubation and experiments with labeled hydroxyurea show the NO derives from the drug. Gas chromatography–mass spectrometry measurements indicate the hydrolysis of hydroxyurea to hydroxylamine in this system. Incubation of hydroxylamine with crude rat liver homogenate also generates nitrite/nitrate and iron nitrosyl complexes. A line of evidence including inhibitor studies, EPR spectroscopy, and nitrite/nitrate detection identifies catalase as a possible oxidant for the conversion of hydroxyurea to NO. These results reveal the ability of liver tissue to convert hydroxyurea to nitric oxide and provide insight into the metabolism of this drug.

The study of ATM/ATR gene expression and cell apoptosis, cell cycle change in K562 cells treated with various HU concentration

To investigate the effects of hydroxyurea on ATM/ATR mRNA expression, corresponding dynamic change of cell cycle and mechanisms of apoptosis. Erythyroleukemia cell line K562 cells were treated with various concentration of hydroxyurea, RT-PCR was introduced to semiquantify the ATM/ATR mRNA expression and flow cytometry was applied to measure the BCL-2, Annexin V and the cell cycle. Increasing the concentration of hydroxyurea resulted in enhanced ATM gene expression while the ATR and BCL-2 decreased. BCL-2 expression positive rate were 38%, 33%, 30%, 28% and 18%. BCL-2 expression was different significantly with 0.25 and 2.0 #muWmol/L hydroxyurea (P<0.05). Neither ATM nor ATR expresses in the untreated K562 cells. Furthermore, more apoptosis and G1 phase cells with decreased S and G2/M phase cells could be also observed. The dynamic change of ATM/ATR gene expression is in a manner of hydroxyurea dose-dependent which is involved in the cell cycle change and cell apoptosis.

Hydroxyurea Induced Expression of Erythrocyte Anion Exchange Protein [AE1] and Gamma Globin in Cultured Erythroid Progenitor Cells.

Background: Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell anemia and beta-thalassemia. The clinical efficacy of HU is due primarily to increases in fetal hemoglobin (HbF) levels. HU increases the %HbF and the %F cells. The HU reactive mechanism(s) in erythroid cells, however, have not been clearly defined. Patients receiving HU therapy develop subpopulations of macrocytic erythrocytes. Our previous studies demonstrate that sickle cell patients treated with low dose HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. The frequency of cells expressing greater levels of (AE1) per cell was increased in each serial blood sample. We propose that part of the HU reactive mechanism will include the upmodulation of non-gamma globin erythroid proteins that contribute to the macrocytic structures. As part of our investigation of the development of RBCs expressing increased band 3 protein per cell, we examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors.Methods: HU induced protein synthesis was investigated as a function of HU concentration. Erythroid progenitors were cultured in serum free semisolid media containing different concentrations of HU [0–40 micromolar]. BFU-E were scored and harvested after 15 days in culture, then assayed. BFU-E derived cells were first labeled with monoclonal anti-band 3 antibody. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry. BFU-E derived cells were subsequently labeled with anti-spectrin antibody plus monoclonal anti-band 3 antibody, then assayed. For the next set of experiments, BFU-E cultured in increasing concentrations of HU were assayed for the presence of band 3 protein and gamma globin. BFU-E cells were fixed permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody, assayed by flow cytometry.Results: Results show that the frequency of cells positive for band 3 protein [AE1] was increased at increasing concentrations of HU as compared to controls. The band-3 upmodulation appears to plateau at 12.5–20 micromolar HU. Band 3 protein derived from BFUE was confirmed by Western blot analysis. The frequency of cells positive for both band 3 and spectrin was similarly increased in cells cultured in the presence of increasing concentrations of HU. In separate studies, cells cultured in HU were assayed for the expression of both gamma globin and AE1 by flow cytometry. Results show a 2–3-fold increase in the % band 3 [AE1] plus gamma globin positive cells in HU [5–40 micromolar] treated cells as compared to untreated controls.Conclusions: These results demonstrate that HU induces the expression of band 3 and gamma globin in cultured erythroid progenitors. These findings suggest that part of the mechanism of HU action in erythroid cells involves the induction of erythroid structural proteins concordant with the induction of gamma globin.

Hydroxyurea Therapy Reduces the Gene Expression of Adhesion Molecules VLA-4 and CD36 in Sickle Erythrocytes, with a Concomitant Decrease in Adhesion to Fibronectin.

Adhesive interactions between sickle red cells, leukocytes and the endothelium are thought to initiate vaso-oclusive events in the microcirculation. An increased expression of adhesion molecules such as CD 36 (thrombospondin receptor) and VLA-4 integrin (α4β1, CD49d/CD29) has been observed on the surface of sickle reticulocytes and the increased adhesive properties of sickle erythrocytes are well documented. Gene expression of adhesion molecules and their modulation by drugs such as hydroxyurea (HU) have yet to be fully explored. We compared the expression of the CD36 gene and the VLA-4 integrin subunit genes, CD29 and CD49d, in the reticulocytes of steady-state SCD patients and patients on HU therapy (20–30 mg/Kg/day) using Real Time PCR and also compared the basal adhesion of erythrocytes from these subjects using static adhesion assays. Basal adhesion of sickle erythrocytes (2x108 cells/ml) to fibronectin (20mg/ml) - coated plates was significantly higher than normal erythrocyte adhesion (15.4 ± 2.8% compared to 5.9 ± 1.1%, n=9, p=0.008). In contrast, the adhesion of erythrocytes from SS patients on HU therapy to fibronectin was significantly lower than that of SS erythrocytes of patients not on HU (5.8 ± 0.7%, n=9, p=0.006). Pre-treatment of cells, in vitro, with varying concentrations of HU (0.1 → 250μM, 30 minutes, 37oC), had no significant effect on either normal or sickle cell erythrocyte adhesion under our experimental conditions, suggesting that HU may not reduce sickle cell adhesion by altering adhesion molecule function due to affinity changes. We next used real time PCR to investigate the expression of the mRNA of the CD36 gene and the genes for the VLA-4 subunits, CD49d and CD29, in the reticulocytes of steady-state SCD patients and patients on HU therapy. CD 36 gene expression was reduced by 62.5% ± 10.4% in the reticulocytes of patients on HU compared to the reticulocytes of SCD patients not on HU (n=8, p < 0.001). The β1 (CD29) integrin subunit expression was reduced by 87.9 ± 8.7% (n=8, p < 0.001) in patients on HU compared to SS reticulocytes; likewise α4 integrin (CD49d), expression was reduced by 50.0% ± 2.6% (n=8, p <0.001). In addition, reticulocyte mRNA was collected from one steady-state SCD patient before and 3 months after initiation of HU therapy and decreases of 99.5%, 98.0% and 98.0% were observed in the expressions of CD36, CD29 and CD49d mRNA, respectively, in the reticulocytes of this patient following HU therapy. We speculate that the decrease in adhesive properties observed in the erythrocytes of sickle cell patients during HU therapy may be the consequence of a down-regulation of the gene expressions of adhesion molecules and, consequently, protein expression by sickle cells rather than as a result of alterations in adhesion molecule function.