Monoterpenes as therapeutic candidates to induce fetal hemoglobin synthesis and up-regulation of gamma-globin gene: An in vitro and in vivo investigation.

Pharmacologically induced production of fetal hemoglobin (HbF) is a pragmatic therapeutic strategy for the reduction of globin chain imbalance and improving the clinical severities of patients with ß-hemoglobinopathies. To identify highly desirable new therapeutic HbF-inducing agents, we screened functionally diverse ten monoterpenes, as molecular entities for their potent induction and erythroid differentiation ability in human erythroleukemia cell line (K562) and transgenic mice. Benzidine hemoglobin staining demonstrated six compounds to have significantly induced erythroid differentiation of K562 cells in a dose and time-dependent manner. This induction paralleled well with the optimal accumulated quantity of total hemoglobin in treated cultures. The cytotoxic studies revealed that three (carvacrol, 3-carene, and 1,4-cineole) of the six compounds with their maximal erythroid expansion ability did not affect cell proliferation and were found non-toxic. Four compounds were found to have high potency, with 4-8-fold induction of HbF at both transcriptional and protein levels in vitro. Subsequently, an in vivo study with the three active non-cytotoxic compounds showed significant overexpression of the γ-globin gene and HbF production. Carvacrol emerged as a lead HbF regulator suggested by the increase in expression of γ-globin mRNA content (5.762 ± 0.54-fold in K562 cells and 5.59 ± 0.20-fold increase in transgenic mice), accompanied by an increase in fetal hemoglobin (F-cells) levels (83.47% in K562 cells and 79.6% in mice model). This study implicates monoterpenes as new HbF inducing candidates but warrants mechanistic elucidation to develop them into potential therapeutic drugs in ß-thalassemia and sickle cell anemia.

Oral Monomethyl Fumarate Therapy Ameliorates Retinopathy in a Humanized Mouse Model of Sickle Cell Disease.

AIMS: Sickle retinopathy (SR) is a major cause of blindness in sickle cell disease (SCD). The genetic mutation responsible for SCD is known, however; oxidative stress and inflammation also figure prominently in the development and progression of pathology. Development of therapies for SR is hampered by the lack of (a) animal models that accurately recapitulate human SR and (b) strategies for noninvasive yet effective retinal drug delivery. This study addressed both issues by validating the Townes humanized SCD mouse as a model of SR and demonstrating the efficacy of oral administration of the antioxidant fumaric acid ester monomethyl fumarate (MMF) in the disease. RESULTS: In vivo ophthalmic imaging, electroretinography, and postmortem histological RNA and protein analyses were used to monitor retinal health and function in normal (HbAA) and sickle (HbSS) hemoglobin-producing mice over a one-year period and in additional HbAA and HbSS mice treated with MMF (15 mg/ml) for 5 months. Functional and morphological abnormalities and molecular hallmarks of oxidative stress/inflammation were evident early in HbSS retinas and increased in number and severity with age. Treatment with MMF, a known inducer of Nrf2, induced γ-globin expression and fetal hemoglobin production, improved hematological profiles, and ameliorated SR-related pathology. Innovation and Conclusion: United States Food and Drug Administration-approved formulations in which MMF is the primary bioactive ingredient are currently available to treat multiple sclerosis; such drugs may be effective for treatment of ocular and systemic complications of SCD, and given the pleiotropic effects, other nonsickle-related diseases in which oxidative stress, inflammation, and retinal vascular pathology figure prominently. Antioxid. Redox Signal. 25, 921-935.

Expression of the significance of silent information regulator type-1 in Angioimmunoblastic T-cell lymphoma is greater association with tumorigenesis and has strong implications for adverse prognosis.

Silent information regulator type-1 (SIRT1) is the best-studied member of the Sirtuin (Sir2) family of nicotinamide dinucleotide (NAD)-dependent class III histone deacetylases (HDACs). Rrecently, it is suggested that SIRT1 may be involved in the development of malignant tumors including mouse lymphoma, but has not yet been explored in Angioimmunoblastic T-cell lymphoma (AITL). Therefore, we investigated the prevalence and the prognostic impact of SIRT1 expression in AITL. Immunohistochemical expression of SIRT1, p53 were evaluated by using a 2 mm core from 45 AITL patients. Positive expression of SIRT1 was seen in 71.11% (32 of 45) of patients and p53 expression were seen in 53.33% (24 of 45). SIRT1 and p53 expression were significantly associated with shorter PFS by univariate analysis (P=0.009 and P < 0.001, respectively), multivariate analysis also shows that SIRT1 expression relate to worse prognosis. We also suggest inferior survival in AITL with the combined expression of SIRT1 and clinical characteristics of high IPI scores, high clinical stage, increased serum LDH, decreased HGB and increased γ-Globulin. In conclusion, our results indicate that SIRT1 is strongly expressed in AITL and it act as a clinically significant prognostic indicator for AITL patients, may also serve as a therapeutic target in AITL.

A phase I, pharmacokinetic, and pharmacodynamic evaluation of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine, administered with tetrahydrouridine.

PURPOSE: Inhibitors of DNA (cytosine-5)-methyltransferases (DNMT) are active antineoplastic agents. We conducted the first-in-human phase I trial of 5-fluoro-2'-deoxycytidine (FdCyd), a DNMT inhibitor stable in aqueous solution, in patients with advanced solid tumors. Objectives were to establish the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of FdCyd + tetrahydrouridine (THU). METHODS: FdCyd + THU were administered by 3 h IV infusion on days 1-5 every 3 weeks, or days 1-5 and 8-12 every 4 weeks. FdCyd was administered IV with a fixed 350 mg/m(2)/day dose of THU to inhibit deamination of FdCyd. Pharmacokinetics of FdCyd, downstream metabolites and THU were assessed by LC-MS/MS. RBC γ-globin expression was evaluated as a pharmacodynamics biomarker. RESULTS: Patients were enrolled on the 3-week schedule at doses up to 80 mg/m(2)/day without dose-limiting toxicity (DLT) prior to transitioning to the 4-week schedule, which resulted in an MTD of 134 mg/m(2)/day; one of six patients had a first-cycle DLT (grade 3 colitis). FdCyd ≥40 mg/m(2)/day produced peak plasma concentrations >1 µM. Although there was inter-patient variability, γ-globin mRNA increased during the first two treatment cycles. One refractory breast cancer patient experienced a partial response (PR) of >90 % decrease in tumor size, lasting over a year. CONCLUSIONS: The MTD was established at 134 mg/m(2) FdCyd + 350 mg/m(2) THU days 1-5 and 8-12 every 4 weeks. Based on toxicities observed over multiple cycles, good plasma exposures, and the sustained PR observed at 67 mg/m(2)/day, the phase II dose for our ongoing multi-histology trial is 100 mg/m(2)/day FdCyd with 350 mg/m(2)/day THU.

Pivotal role of HIV and EBV replication in the long-term persistence of monoclonal gammopathy in patients on antiretroviral therapy.

A high prevalence of monoclonal gammopathy (MG) has been observed in HIV-infected patients. We explored the conditions associated with long-term persistence of serum monoclonal protein (M protein) in HIV-infected patients on antiretroviral therapy (ART). Of 21 patients with MG, M protein disappeared in 12 patients (58%) over 5 years of ART. Higher level of serum γ-globulin and higher percentages of circulating plasmablasts and plasma cells were observed in patients with persistent MG compared with patients with transient MG. MG persistence was associated with the cumulative time of detectable plasma HIV RNA after ART initiation, detection of Epstein-Barr virus (EBV) DNA in plasma, and a high level of EBV DNA in B cells. Poor control of HIV replication and detectable EBV replication in plasma were both associated with long-term MG persistence in patients on ART. In the case of viral control, MG associated with HIV infection is usually transient.

Molecular pharmacological basis of the YiSui ShenXu Granule in beta-thalassemia therapy.

OBJECTIVES: To study the molecular pharmacological basis of the YiSui ShenXu Granule, a complex prescription of the Chinese traditional medicine used to treat beta-thalassemia. METHODS: Real-time quantitative PCR method had been applied to analyze the genes expression: gamma-globin, Ckit, EpoR, Spi, FKLF, GATA1 and GATA2 in K562 cell treated and untreated with this complex prescription and its each single herbal medicine. RESULTS: The results showed that this complex prescription increased the gamma-globin, EpoR, Spi and FKLF expression and decreased the Ckit, GATA1 and GATA2 expression. And all single herbal medicines of this complex prescription could change some of those gene expressions, but not the same as the complex prescription. Even that, this study results indicated that the YiSui ShenXu Granule has its molecular pharmacological basis in treating beta-thalassemia.

Thalidomide induces gamma-globin gene expression through increased reactive oxygen species-mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis.

Although thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide's specific effects on gamma-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on gamma-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34+ cells. We found that thalidomide induced gamma-globin mRNA expression in a dose-dependent manner, but had no effect on beta-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogen-activated protein kinase (MAPK) signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. Pretreatment of cells with the antioxidant enzyme catalase and the intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide-induced p38 MAPK activation and histone H4 acetylation. Moreover, pretreatment with catalase and DMTU diminished thalidomide-induced gamma-globin gene expression. These data indicate that thalidomide induces increased expression of the gamma-globin gene via ROS-dependent activation of the p38 MAPK signaling pathway and histone H4 acetylation.

Selection with a regulated cell growth switch increases the likelihood of expression for a linked gamma-globin gene.

Several lines of evidence indicate that in vivo drug selection can be used to overcome the low rates of gene transfer and engraftment encountered in many hematopoietic stem cell gene therapy settings. However, whether selection imposed on one transcription cassette effects the likelihood of expression from a second, independent transcription cassette within the same vector has been less well studied. In order to address this issue, we engineered an oncoretrovirus vector to express two separate transcription units: (i) a bicistronic cassette encoding both GFP and a pharmacologically regulated cell growth switch based on the thrombopoietin receptor Mpl; and (ii) a highly position-dependent second cassette encoding human gamma-globin. Studies in cell cultures and in mice transplanted with transduced marrow indicated that selective expansion increased by more than 9-fold the fraction of erythroid cells expressing the linked but separate expression cassette for gamma-globin. This increase was far greater then that observed for the bicistronic GFP gene, and cannot be explained by a simple increase in the fraction of cells containing provirus. These results suggest that selective expansion favors erythroid stem/progenitor cells with provirus integrated at chromosomal sites which are relatively resistant to silencing position effects.

High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors.

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.

Development of a stable retrovirus vector capable of long-term expression of gamma-globin mRNA in mouse erythrocytes.

Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human gamma-globin gene showed position-independent, copy number-dependent expression of a linked gamma-globin mRNA. We generated a "double-copy" Ank/A gamma-globin retrovirus vector that transferred two copies of the Ank/A gamma-globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long-term repopulating hematopoietic stem cells. Expression of Ank/A gamma-globin mRNA in mature red blood cells was approximately 8% of the level of mouse alpha-globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.

Expression of erythroid-specific genes in megakaryoblastic disorders.

Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other, and there may exist progenitor cells common to those two lineages may exist. Acute megakaryoblastic leukemia (AML-M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase (ALAS-E), indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages.

Greater secretion of growth hormone in black than in white men: possible factor in greater bone mineral density--a clinical research center study.

To determine why blacks have a higher bone mineral density (BMD) and lower incidence of osteoporosis and fractures than whites, we investigated whether the secretion of GH is higher in black than in white men. Measurements of GH were obtained at 20-min intervals over 24 h and analyzed by deconvolution. BMD was determined by dual energy x-ray absorptiometry in 16 normal black and 17 normal white men, aged 20-40 yr. The 24-h integrated GH concentration 942 +/- 174 vs. 602 +/- 104 micrograms/L; P = 0.0495) and GH secretory burst amplitude (0.499 +/- 0.163 vs. 0.169 +/- 0.027 micrograms/L.min; P = 0.0482) were higher in black than in white men. GH burst frequency, half-duration, mass, and half-life were not different in the 2 groups. The serum 17 beta-estradiol level (162 +/- 12 vs. 108 +/- 11 pmol/L; P = 0.0011) was higher, and the serum insulin-like growth factor-binding protein 3 level (2.2 +/- 0.1 vs. 2.8 +/- 0.1 microgram/mL; P = 0.0001) was lower in black than in white men. BMD values for total body (1.22 +/- 0.02 vs. 1.14 +/- 0.02 g/cm2; P = 0.0041), forearm (0.69 +/- 0.01 vs. 0.66 +/- 0.01 g/cm2; P = 0.0211), trochanter (0.91 +/- 0.03 vs. 0.77 +/- 0.03 g/cm2; P = 0.0003), and femoral neck (1.08 +/- 0.03 vs. 0.93 +/- 0.03 g/cm2; P = 0.0007) were higher in black than in white men. Thus, serum 17 beta-estradiol level, GH secretion, and BMD values for the total body, forearm, trochanter, and femoral neck are greater in black than in white men. As estrogen is known to increase GH secretion and GH to increase bone mass, increases in circulating 17 beta-estradiol may contribute to the higher GH secretion and bone mass in black men.

Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines.

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.

[Antigenic composition of serum proteins of the Inv system in normal conditions and in patients with hematologic diseases among the Armenian population]. / Antigennyi sostav syvorotochnykh belkov sistemy Inv v norme i pri gematologicheskikh zabolevaniiakh v populiatsii armian.

Inv (1) antigen distribution was studied in 568 normal subjects and in 354 hematological patients in the Armenian population. Inv (1) antigen was detected in 16.7% of the normal Armenians studied. The incidence rate of Inv (1) factor does not depend on the distribution of phenotypes of ABO system, rhesus factor (D), and the sex of the subjects investigated. Inv (1) antigen incidence rate in patients with acute leukemia, chronic lymphocytic leukemia, iron deficiency anemia, lymphogranulomatosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia was similar to that in the control, and only patients with chronic myeloid leukemia had significantly decreased levels of Inv (1) antigen: 6.8% as compared to 16.7% in the population.

Frequent occurrence of Ha-rasl allelic deletion in human ovarian adenocarcinomas.

Fourteen human adenocarcinoma specimens were analyzed for somatic abnormalities affecting genes of the ras family. No amplification of the 3 ras genes was detected. Allelic deletion of the Ha-rasl gene (11p15.5) was found to be a very common abnormality in human ovarian adenocarcinomas (4 out of 7 informative cases). However, in these neoplasm deletion of a presumed normal Ha-rasl allele is not a contributory factor in strengthening the tumorigenic effect of a mutated allele. More probably, Ha-rasl allelic losses are markers of larger chromosomal deletions. Analyses at gamma globin loci (11p15.5) and int-2 locus (11q13) provided evidence that the deletions may extend from Ha-rasl locus towards the centromere but never involve loss of the entire chromosome 11. These findings may suggest that a putative tumor suppressor gene closely linked to Ha-rasl in 11p15.5 is involved in ovarian cancerogenesis.

Autocrine effect of endothelin on DNA synthesis in human vascular endothelial cells.

To elucidate the role of endogenous endothelin on DNA synthesis in endothelial cells, we have investigated the effects of rabbit anti-ET gamma globulin on DNA synthesis in cultured human vascular endothelial cells. DNA synthesis was markedly inhibited by anti-ET gamma globulin in a concentration dependent manner in the presence of fetal calf serum(FCS) (x5000;34%, x2500;54%, x1000;70%), but not in the absence of FCS. These data suggest that endogenous ET modulates FCS-stimulated DNA synthesis in an autocrine fashion.

Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells.

We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated "A gamma-neo" transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

Regulation of human globin gene expression in mouse erythroleukemia x human fibroblast hybrid cells.

A somatic cell hybrid, XX-8, was obtained from a fusion of tetraploid mouse erythroleukemia cells with human Lesch-Nyhan skin fibroblasts. This hybrid cell was previously shown (1) to produce human beta- but no human gamma-globin mRNA sequences after induction with dimethylsulfoxide. In this study we show that: (a) human beta- and gamma-globin genes are present in XX-8 cells in approximately equal numbers; (b) no human gamma-globin mRNA sequences can be detected in either the cytoplasmic or nuclear RNA fractions even with several different inducers; (c) after induction the human beta-globin gene is converted from a DNase I insensitive or closed structure to a DNase I open configuration, while the human gamma-globin gene remains closed; and (d) no human beta-globin polypeptide can be detected in the intact induced cells, indicating that fibroblast globin genes, even when induced to make mRNA in an erythroid environment, do not synthesize an RNA that is translated efficiently.