Gordon D. Ginder

A short-term trial of butyrate to stimulate fetal-globin-gene expression in the β-globin disorders

BACKGROUND Fetal-globin (gamma-globin) chains inhibit the polymerization of hemoglobin S (sickle hemoglobin) and can functionally substitute for the beta-globin chains that are defective or absent in patients with the beta-thalassemias. Identifying safe mechanisms to stimulate fetal-hemoglobin production is therefore of great interest. Previous studies have shown that administering butyrate selectively stimulates the promoter of the human fetal-globin gene and leads to increases in gamma-globin--gene expression in the developing fetus, cultured cells, and animal models. METHODS To determine whether butyrate can stimulate fetal-globin production in humans, we treated three patients (3 to 13 years old) with sickle cell anemia and three patients (7 to 27 years old) with beta-thalassemia syndromes with a short course of intravenous infusions of arginine butyrate. The drug was infused continuously for either two or three weeks; the initial dose was 500 mg per kilogram of body weight per day. Globin-chain ratios, proportions of reticulocytes producing hemoglobin F (F reticulocytes), and levels of gamma-globin messenger RNA (mRNA) were determined before and during treatment. RESULTS In all six patients, fetal-globin synthesis increased by 6 to 45 percent above pretreatment levels (P < 0.01). The proportion of F reticulocytes increased about twofold, and the level of gamma-globin mRNA increased twofold to sixfold. The increase in gamma-globin synthesis led to improvement in the globin-chain ratios in the patients with thalassemia. The treatment of one patient was extended for seven weeks, and her hemoglobin level increased from 4.7 to 10.2 g per deciliter (2.9 to 6.3 mmol per liter). Side effects were minimal; one patient had a transient increase in serum aminotransferase concentrations. CONCLUSIONS In patients with beta-hemoglobinopathies butyrate, a natural fatty acid, can significantly and rapidly increase fetal-globin production to levels that can ameliorate beta-globin disorders. Further trials of this class of compounds are warranted to determine long-term tolerance and efficacy in patients with sickle cell anemia or beta-thalassemia.

Extended Therapy with Intravenous Arginine Butyrate in Patients with β-Hemoglobinopathies

BACKGROUND Enhanced production of fetal hemoglobin lessens the severity of beta-thalassemia and sickle cell disease. Intravenous infusion of arginine butyrate can increase the number of reticulocytes containing fetal hemoglobin in patients with these disorders, and it has induced a substantial increase in hemoglobin in one patient with thalassemia. We therefore tested the efficacy of this agent in patients with beta-hemoglobinopathies. METHODS We treated 10 patients with severe beta-thalassemia or sickle cell disease with arginine butyrate at an initial dose of 500 mg per kilogram of body weight per day (final dose, 2000 mg per kilogram per day), 6 days per week, for a mean (+/- SD) of 10 +/- 1.2 weeks (range, 9 to 13). A hematologic response was defined as an increase in the hemoglobin concentration of at least 2 g per deciliter in patients with thalassemia and as a twofold increase in the fetal hemoglobin concentration in patients with sickle cell disease. RESULTS Increase in gamma-globin messenger RNA and in reticulocytes containing fetal hemoglobin but not in hemoglobin were observed in the patients with thalassemia. A small, unsustained increase in fetal hemoglobin was observed in two patients with sickle cell disease. Drug toxicity was minimal at standard doses. One patient had a grand mal seizure after inadvertently receiving 2000 mg of arginine butyrate per kilogram over a period of six hours. CONCLUSIONS Ten weeks of intravenous arginine butyrate did not produce a hematologic response in 10 patients with either severe beta-thalassemia or sickle cell disease.

Treatment of thalassaemia major with phenylbutyrate and hydroxyurea

Vol 350 • August 16, 1997 491 reference to the drug-free healthy volunteer group. Drug-treated groups had reduced binding in all areas compared with healthy volunteers (p<0·0001). A significant difference between clozapine and typical antipsychotic-treated groups was found bilaterally in the striatum (p=0·005). Clozapine-treated patients showed significantly lower occupancy of striatal D 2 /D 3 receptors than typical antipsychotic-treated patients (58 [SE 3] vs 90 [SE 3]; mean percent occupancy for clozapine and typical antipsychotictreated groups, respectively); this finding is fully consistent with previous data. There were no differences in the temporal cortex between clozapine and typical antipsychotic groups. Mean percent occupancy of D 2 /D 3 receptors in this region was very high in both cases (90 [SE 3] and 96 [SE 2] for clozapine and typical antipsychotic groups, respectively; see figure). The differential action of clozapine, whereby it blocks extrastriatal dopamine D 2 /D 3 receptors in excess of striatal receptors, provides an important explanation for its clinical profile. Drug discovery for new antipsychotics should concentrate on highly selective D 2 blockers with affinity at dopamine D 2 /D 3 receptors similar to clozapine.

Elimination of Transfusions Through Induction of Fetal Hemoglobin Synthesis in Cooley's Anemiaa

Pharmacological stimulation of fetal hemoglobin production is of considerable interest as an alternative approach to therapy for Cooleys anemia. While intravenous compounds have been effective in inducing short‐term increases in fetal hemoglobin in a few patients, long‐term elimination of transfusion requirement has not been reported. In patients with Cooleys anemia, treatment with oral sodium phenylbutyrate alone, sodium phenylbytyrate combined with hydroxyurea, and hydroxyurea alone, has augmented fetal hemoglobin production and increased total hemoglobin concentration as much as 5 g/dl over baseline eliminating transfusion requirement in two patients. Parallel declines in circulating nucleated red cell count, and concentrations of serum transform receptor and erythropoietin. are consistent with more effective erythropoiesis. Over extended periods of treatment, no induction of other fetal proteins and no adverse effects were observed. Particular disease mutations and other genetic factors may be of prime importanec in determining the response to agents that induce production of fetal hemoglobin.

Interferon-γ Induction of the Human Leukocyte Antigen-E Gene Is Mediated through Binding of a Complex Containing STAT1α to a Distinct Interferon-γ-responsive Element

Expression of the human major histocompatibility complex (MHC) class I genes has been shown previously to increase at the transcriptional level following exposure to interferon-γ (IFN-γ). In this report we have examined the molecular mechanisms involved in the IFN-γ-induced transcription of the human MHC class I gene, HLA-E. Functional analysis of CAT reporter gene constructs under the control of the HLA-E promoter transfected into U937 cells revealed the presence of a distinct IFN-γ-responsive element, termed the interferon response region (IRR), that was necessary and sufficient to mediate the response to IFN-γ. This cis-acting regulatory sequence contains an imperfect inverted repeat; the 5′-half of the IRR resembles the IFN-γ activation site (GAS), and the 3′-half of the IRR resembles the interferon-stimulated response element (ISRE). Gel mobility shift assays demonstrated that the IRR bound a single, specific, IFN-γ-induced complex (IRR-AC), which was formed rapidly following treatment with IFN-γ and was independent of protein synthesis. Competition experiments with GAS and ISRE sequences from other IFN-inducible genes showed that GAS sequences competed for the IRR-AC, whereas ISRE sequences did not compete. Mutational analysis demonstrated that point mutations in either the 5′-half or 3′-half of the IRR prevented binding of the complex and abrogated or markedly reduced the IFN-γ responsiveness of reporter gene constructs. Supershift analysis revealed that the IRR-AC contains a factor that was recognized by antibodies specific for the protein STAT1α (signal transducer and activator of transcription). Taken together, these findings suggest that the mechanism of IFN-γ-induced transcription of the HLA-E gene is distinct from that of other MHC class I genes.

Differential IFN-γ Stimulation of HLA-A Gene Expression through CRM-1-Dependent Nuclear RNA Export

IFNs regulate most MHC class I genes by stimulating transcription initiation. As shown previously, IFN-γ controls HLA-A expression primarily at the posttranscriptional level. We have defined two 8-base sequences in a 39-nucleotide region in the 3′-transcribed region of the HLA-A gene that are required for the posttranscriptional response to IFN-γ. Stimulation of HLA-A expression by IFN-γ requires nuclear export of HLA-A mRNA by chromosome maintenance region 1 (CRM-1). Treatment of cells with leptomycin B, a specific inhibitor of CRM-1, completely inhibited IFN-γ induction of HLA-A. Expression of a truncated, dominant-negative form of the nucleoporin NUP214/CAN, ΔCAN, that specifically interacts with CRM-1, also prevented IFN-γ stimulation of HLA-A, providing confirmation of the role of CRM-1. Increased expression of HLA-A induced by IFN-γ also requires protein methylation, as shown by the fact that treatment of SK-N-MC cells or HeLa cells with the PRMT1 inhibitor 5′-methyl-5′-thioadenosine abolished the cellular response to IFN-γ. In contrast with HLA-A, IFN-γ-induced expression of the HLA class Ib gene, HLA-E, was not affected by either 5′-methyl-5′-thioadenosine or leptomycin B. These results provide proof of principle that it is possible to differentially modulate the IFN-γ-induced expression of the HLA-E and HLA-A genes, whose products often mediate opposing effects on cellular immunity to tumor cells, pathogens, and autoantigens.

Identification of CCAAT displacement protein (CDP/cut) as a locus-specific repressor of major histocompatibility complex gene expression in human tumor cells.

Human major histocompatibility (MHC) class I antigen expression is important in controlling the metastatic growth of malignant tumors. Locus-specific down-regulation of MHC class I gene expression is frequently observed in human tumors, leading to decreased susceptibility to cytotoxic T-cell-mediated lysis. The mechanism of this down-regulation is incompletely understood. Here, we describe the identification of human CCAAT displacement protein (CDP/cut) as a locus-specific repressor of HLA-B and C gene expression. Transient and stable transfections in HeLa and K562 cells demonstrated the presence of a repressor element 650 base pairs upstream of the first exon of HLA-B7. A specific binding complex with the HLA-B7 and Cw2 repressor elements was demonstrated by EMSA. Formation of the EMSA complex was inhibited specifically with polyclonal antiserum to human CDP/cut, demonstrating that CDP/cut binds the HLA-B7 repressor element. The corresponding region of the HLA-A2 promoter neither repressed HLA-A2 gene expression nor bound CDP/cut. Overexpression of CDP/cut in cell lines deficient in CDP/cutresulted in a nearly 4-fold repression of reporter constructs containing the HLA-B7 repressor element but not the corresponding region of the HLA-A2 promoter. Repression of HLA-B and C gene expression by CDP/cut does not involve displacement of NF-Y, nor is CDP/cut associated with the histone deacetylase HDAC1 when bound to the HLA-B7 repressor element. To our knowledge, these results identify CDP/cut as the first example of a locus-specific repressor of MHC class I gene transcription in human tumor cells.

A 3′-Transcribed Region of the HLA-A2 Gene Mediates Posttranscriptional Stimulation by IFN-γ

The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-γ. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-γ stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5′ IFN-stimulated response element consensus sequence, is not sufficient for IFN-γ response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5′- and 3′-flanking sequences, resulted in stimulation of the gene by IFN-γ. Nuclear run-on assays revealed that, unlike other class I genes, IFN-γ stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3′ end was unaffected by IFN-γ treatment. Sequences that mediate the majority of IFN-γ induction of HLA-A2 mRNA reside in a 127-bp 3′-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-γ treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-γ.