Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-κB pathways in human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Diabetes mellitus (DM) is a risk factor for periodontal diseases and may exacerbate the progression of the pathogenesis of periodontitis. Advanced glycation end-products (AGEs) cause DM complications relative to levels of glycemic control and larger amounts accumulate in the periodontal tissues of patients with periodontitis and DM. In the present study, we investigated the effects of AGEs on the expression of inflammation-related factors in human gingival fibroblasts (HGFs) to elucidate the impact of AGEs on DM-associated periodontitis. MATERIAL AND METHODS: HGFs were cultured with or without AGEs. Cell viability was examined, and RNA and protein fractions were isolated from AGE-treated cells. The expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), and the receptor for AGE (RAGE) was investigated using reverse transcription-polymerase chain reaction, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and reactive oxygen species activity was measured using a kit with 2',7'-dichlorofluorescin diacetate. Human monocytic cells (THP-1) labeled with a fluorescent reagent were co-cultured with HGFs treated with AGEs and IL-6 siRNA, and the adhesive activity of THP-1 cells to HGFs was assessed. The expression of IL-6 and ICAM-1 was examined when HGFs were pretreated with recombinant human IL-6, the siRNAs of RAGE and IL-6, and inhibitors of MAPK and NF-κB, and then cultured with and without AGEs. The phosphorylation of MAPK and NF-κB was assessed using western blotting. RESULTS: AGEs increased the mRNA and protein expressions of RAGE, IL-6, ICAM-1 and reactive oxygen species activity in HGFs, and promoted the adhesion of THP-1 cells to HGFs, but had no effect on cell viability until 72 hours. Recombinant human IL-6 increased ICAM-1 expression in HGFs, while the siRNAs of RAGE and IL-6 inhibited AGE-induced IL6 and ICAM1 mRNA expression, and IL-6 siRNA depressed AGE-induced THP-1 cell adhesion. AGEs increased the phosphorylation of p38 and ERK MAPKs, p65 NF-κB and IκBα, while inhibitors of p38, ERK MAPKs and NF-κB significantly decreased AGE-induced IL-6 and ICAM-1 expression. CONCLUSION: AGEs increase IL-6 and ICAM-1 expression via the RAGE, MAPK and NF-κB pathways in HGFs and may exacerbate the progression of the pathogenesis of periodontal diseases.

Coping strategies and risk of cardiovascular disease incidence and mortality: the Japan Public Health Center-based prospective Study.

AIMS: Coping strategies may be significantly associated with health outcomes. This is the first study to investigate the association between baseline coping strategies and cardiovascular disease (CVD) incidence and mortality in a general population cohort. METHODS AND RESULTS: The Japan Public Health Center-based prospective Study asked questions on coping in its third follow-up survey (2000-04). Analyses on CVD incidence and mortality included 57 017 subjects aged 50-79 without a history of CVD and who provided complete answers on approach- and avoidance-oriented coping behaviours and strategies. Cox regression models, adjusted for confounders, were used to determine hazard ratios (HRs) according to coping style. Mean follow-up time was 7.9 years for incidence and 8.0 years for mortality.The premorbid use of an approach-oriented coping strategy was inversely associated with incidence of stroke (HR = 0.85; 95% CI, 0.73-1.00) and CVD mortality (HR = 0.74; 95% CI, 0.55-0.99). Stroke subtype analyses revealed an inverse association between the approach-oriented coping strategy and incidence of ischaemic stroke (HR = 0.79; 95% CI, 0.64-0.98) and a positive association between the combined coping strategy and incidence of intra-parenchymal haemorrhage (HR = 2.03; 95% CI, 1.01-4.10). Utilizing an avoidance coping strategy was associated with increased mortality from ischaemic heart disease (IHD) only in hypertensive individuals (HR = 3.46; 95% CI, 1.07-11.18). The coping behaviours fantasizing and positive reappraisal were associated with increased risk of CVD incidence (HR = 1.24; 95% CI, 1.03-1.50) and reduced risk of IHD mortality (HR = 0.63; 95% CI, 0.40-0.99), respectively. CONCLUSION: An approach-oriented coping strategy, i.e. proactively dealing with sources of stress, may be associated with significantly reduced stroke incidence and CVD mortality in a Japanese population-based cohort.

Inhibitory effects of advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide on the expression of osteoblastic markers of rat bone marrow cells in culture.

BACKGROUND AND OBJECTIVES: Diabetes is a major risk factor for periodontitis and there is a close relationship between the degree of hyperglycemia and the severity of periodontitis. Advanced glycation end-products (AGEs) accumulate in various tissues under diabetic conditions. AGEs in the periodontal tissues probably play a role in upregulating periodontal inflammation; however, the association of AGEs with the severity of periodontitis has not been fully clarified. Lipopolysaccharide from Porphyromonas gingivalis (P-LPS) is a potent pathogenic factor in periodontitis. Although the independent effect of AGE or P-LPS on osteoblastic cells has been reported in vitro, the effect of adding both has not been clearly elucidated. In this study, to explore factors aggravating diabetic periodontitis, we investigated the effects of AGE and P-LPS on the expression of osteoblastic markers and the expression of inflammation-related markers in vitro. MATERIAL AND METHODS: Rat bone marrow cells were cultured, and alkaline phosphatase activity and bone nodule formation were evaluated as osteoblastic markers. Reverse transcription-polymerase chain reaction was performed to determine the mRNA expression of molecules associated with bone and inflammation. Protein levels of osteocalcin and interleukin-1ß (IL-1ß) were measured using enzyme-linked immunosorbent assay. RESULTS: AGEs and P-LPS independently reduced alkaline phosphatase activity and bone nodule formation. The addition of both AGE and P-LPS (AGE+P-LPS) further decreased these markers. Reverse transcription-polymerase chain reaction analysis revealed that AGE+P-LPS markedly decreased the mRNA expression of osteoblast-related molecules such as type 1 collagen, osteocalcin and Cbfa1, and markedly increased that of inflammation-related molecules such as IL1ß and S100A8. AGE and P-LPS decreased the protein level of osteocalcin and increased that of IL-1ß, and a further increase of IL-1ß was detected for AGE+P-LPS. CONCLUSION: AGEs and P-LPS inhibited the expression of osteoblastic markers and increased the levels of inflammatory markers in rat bone marrow cells, suggesting that both AGE and P-LPS may be important factors associated with the aggravation of diabetic periodontitis.

Inadequate management for secondary fracture prevention in patients with distal radius fracture by trauma surgeons.

UNLABELLED: We evaluated the secondary fracture prevention in 1445 patients with distal radius fracture by trauma surgeons. The rate of patients with distal radius fracture who underwent bone mineral density (BMD) examination was low, suggesting that appropriate treatment for osteoporosis by trauma surgeons is not performed at present. INTRODUCTION: To clarify the status of osteoporosis interventions after distal radial fractures by trauma surgeons who play the main role in treatment for these fractures, we performed a survey involving multiple institutions in Japan. METHODS: We asked 155 board members of the Japanese Society for Fracture Repair for their cooperation and performed a survey in 48 institutions with which members who gave cooperation were affiliated. The subjects consisted of consecutive patients with distal radial fractures occurring between January and December 2012. The presence or absence of a diagnosis of osteoporosis and bone mineral density examination after fracture was investigated. RESULTS: A total of 1445 patients with distal radial fractures were evaluated in this study. BMD examination for diagnosis and treatment for osteoporosis after fracture was performed respectively in 126 (8.7 %) and 193 (13.4 %) of 1445 patients. Treatment for osteoporosis was performed in 93 (73.8 %) of 126 patients who underwent BMD examination after fracture and 100 (8.2 %) of 1219 who did not undergo BMD examination. Of the 126 patients who underwent BMD examination after fracture, 89 showed a BMD <80 % of the young adult mean as a criterion for the initiation of drug treatment for osteoporosis in Japan and 77 (86.5 %) of the 89 patients were treated with drugs. CONCLUSIONS: The rate of patients with distal radial fractures who underwent BMD examination was low, suggesting that appropriate treatment for osteoporosis by trauma surgeons is not performed at present.

The SORL1 gene and convergent neural risk for Alzheimer's disease across the human lifespan.

Prior to intervention trials in individuals genetically at-risk for late-onset Alzheimer's disease, critical first steps are identifying where (neuroanatomic effects), when (timepoint in the lifespan) and how (gene expression and neuropathology) Alzheimer's risk genes impact the brain. We hypothesized that variants in the sortilin-like receptor (SORL1) gene would affect multiple Alzheimer's phenotypes before the clinical onset of symptoms. Four independent samples were analyzed to determine effects of SORL1 genetic risk variants across the lifespan at multiple phenotypic levels: (1) microstructural integrity of white matter using diffusion tensor imaging in two healthy control samples (n=118, age 18-86; n=68, age 8-40); (2) gene expression using the Braincloud postmortem healthy control sample (n=269, age 0-92) and (3) Alzheimer's neuropathology (amyloid plaques and tau tangles) using a postmortem sample of healthy, mild cognitive impairment (MCI) and Alzheimer's individuals (n=710, age 66-108). SORL1 risk variants predicted lower white matter fractional anisotropy in an age-independent manner in fronto-temporal white matter tracts in both samples at 5% family-wise error-corrected thresholds. SORL1 risk variants also predicted decreased SORL1 mRNA expression, most prominently during childhood and adolescence, and significantly predicted increases in amyloid pathology in postmortem brain. Importantly, the effects of SORL1 variation on both white matter microstructure and gene expression were observed during neurodevelopmental phases of the human lifespan. Further, the neuropathological mechanism of risk appears to primarily involve amyloidogenic pathways. Interventions targeted toward the SORL1 amyloid risk pathway may be of greatest value during early phases of the lifespan.

YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes.

OBJECTIVE: YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. SUBJECTS AND METHODS: The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. RESULTS: YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. CONCLUSIONS: GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis.

[Exposure for harvesting internal mammary artery grafts].

Inflated lungs often disturb the harvesting of internal mammary artery grafts. We employ a Universal Stabilizer Arm and Aortic Valve Assistant (Estech Corporation, Danville) to push the mediastinal pleura and the lung away from the upper part of the mediastinum. This system can be attached to any type of retractor and has excellent flexibility. Its use provides good exposure for harvesting the internal mammary artery.

Parry-Romberg syndrome with a clinically silent white matter lesion.

We performed a detailed neuroimaging study in a patient with Parry-Romberg syndrome. Proton MR spectroscopy demonstrated normal spectral patterns, though conventional MR imaging revealed high-intensity areas in the entire white matter in the left hemisphere. Single-photon emission tomography showed increased perfusion in the cortex of the affected hemisphere. Pyramidal tracts and optic radiations were preserved on diffusion tensor tractography. We will correlate these neuroimaging findings with normal psychomotor development in our patient.

Cholesterol sulfate, a second messenger for the eta isoform of protein kinase C, inhibits promotional phase in mouse skin carcinogenesis.

Cholesterol sulfate is a second messenger for the eta isoform of protein kinase C mediating squamous differentiation. We found that cholesterol sulfate inhibited the promotional phase of skin carcinogenesis in female CD-1 mice, which was initiated by 100 micrograms 7,12-dimethylbenz[a]-anthracene and promoted by a single application of 10 micrograms 12-O-tetradecanoylphorbol-13-acetate, followed by repeated applications of 10 micrograms mezerein once a week for 19 weeks. Cholesterol sulfate, when applied topically at a dose of 400 micrograms (820 mumol) 10 min before treatment with the promoters, markedly suppressed tumor formation, resulting in decrease of 56% in the incidence of tumor-bearing mice, 81% in the number of tumors/mouse, and 60% in the size of tumors at 20 weeks of the promotion. This inhibition was not due to elimination of the initiated cells. Treatment with the parental cholesterol at a dose of 320 micrograms (820 mumol), which does not activate the eta isoform, did not inhibit tumor promotion. Repeated treatment with cholesterol sulfate induced scaling of skin at the site of application. Cholesterol sulfate, unlike most inhibitors of tumor promotion, did not inhibit induction of ornithine decarboxylase and hyperplasia in mouse epidermis caused by topical treatment with 12-O-tetradecanoylphorbol-13-acetate. These findings suggest that cholesterol sulfate inhibits tumor promotion by stimulating a differentiation pathway mediated by the eta isoform of protein kinase C.

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal.

Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined to a region spanning nine amino acid residues (positions 561 - 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these findings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.

Growth enhancement of normal human keratinocytes by the antisense oligonucleotide of retinoblastoma susceptibility gene.

We have found that the growth of normal human keratinocytes, grown in serum-free medium, was significantly stimulated by the antisense oligonucleotide of retinoblastoma susceptibility gene (Rb). Normal human keratinocytes were exposed to phosphorothionate oligonucleotides which were complementary to translation initiation codon of Rb gene. The growth of keratinocytes was enhanced by the antisense, but not the sense, oligonucleotide of Rb gene in a dose-dependent manner from 1 to 10 microM. The Rb antisense oligonucleotide, however, did not result in any appreciable change in transcription of the gene when examined by reverse-transcription polymerase chain reaction (RT-PCR) analysis or in the protein expression and the phosphorylation pattern when examined by immunoprecipitation and Western blotting.

Purification and immunological determination of alpha 2-macroglobulin in serum from injured rats.

Rat alpha 2-macroglobulin was isolated and purified from the pooled sera of turpentine-injected rats by sequential use of dextran sulphate, DEAE-cellulose and gel filtration chromatography. The final protein product obtained by this procedure proved to be alpha 2-macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The alpha 2-macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat alpha 2-macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170,000-dalton alpha 2-macroglobulin subunit. The concentration of alpha 2-macroglobulin in the sera of rats was measured by electroimmuno assay a monospecific antiserum against alpha 2-macroglobulin. Purified alpha 2-macroglobulin was used as a standard. Sera from normal male rats contained 32 +/- 4 micrograms of alpha 2-macroglobulin per ml. To determine the time course of response of alpha 2-macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of alpha 2-macroglobulin increased rapidly, reaching a maximum value of 110-140 times that of the control value by 24 h. Little difference was noted in responsiveness between the two sexes.

Cholesterol sulfate activates transcription of transglutaminase 1 gene in normal human keratinocytes.

Cholesterol sulfate and transglutaminase 1 are essential for the process of keratinization. Cholesterol sulfate is formed during keratinization and activates the eta isoform of protein kinase C. Transglutaminase 1 is a key enzyme for formation of the cornified envelope in terminally differentiated keratinocytes. In this study, we demonstrated that cholesterol sulfate acts as a transcriptional activator of the transglutaminase 1 gene in normal human keratinocytes. Growth of normal human keratinocytes was inhibited by cholesterol sulfate, but not by its parental cholesterol. Treatment of normal human keratinocytes with cholesterol sulfate induced activity of transglutaminase 1 in a dose- and time-dependent manner. Activation of transcription of transglutaminase 1 by cholesterol sulfate was demonstrated by northern blotting analysis, whereas that by cholesterol was not. In order to identify a cholesterol sulfate responsive region in the transglutaminase 1 gene, plasmids were constructed containing a luciferase reporter gene ligated to deletion fragments of the 5' upstream region of the tranglutaminase 1 gene and were transfected into normal human keratinocytes. Transfected cells were treated with cholesterol sulfate, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and a high concentration of Ca2+. Our results indicate that the responsive element(s) for cholesterol sulfate and phorbol ester is located upstream of the human transglutaminase 1 gene at a position(s) between -819 and -549, whereas the responsive element for Ca2+ is located at a position between -79 and -49.

Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer.

Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

Structural analysis of mouse tenascin-X: evolutionary aspects of reduplication of FNIII repeats in the tenascin gene family.

Tenascin-X (TNX) is an extracellular matrix glycoprotein involved in both primary structural functions and modulating cellular activities in multicellular organisms. We determined the 67977bp nucleotide sequence of the entire mouse tenascin-X (Tnx) gene, which also includes the last exon of Creb-rp and Cyp21. We compared it with the orthologous human locus. Conservation of both position and orientation of the three functionally unrelated genes at this position was found. Comparison also revealed that introns 1, 4 and 6 of Tnx are highly conserved between species. The sequence showed that mouse Tnx contains 43 exons separated by 42 introns. The deduced amino-acid sequence (4114 residues) revealed that mouse Tnx has a primary structure characteristic of tenascins, which consists of a signal peptide and four heptad repeats followed by 18.5 epidermal growth factor-like (EGF) repeats, 31 fibronectin type III-like (FNIII) repeats, and a region homologous to fibrinogen. cDNA clones generated by alternative splicing of eight consecutive FNIII repeats (M15-M22) as well as a proximal FNIII repeat (M3) were also identified. The FNIII motifs that were subject to alternative splicing were assigned to the group of recently reduplicated FNIII repeats because they have a high level of amino-acid sequence similarity. We also analyzed the evolution of FNIII repeats in TNX.

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1.

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.

UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily.

A selective PKC inhibitor, UCN-01, was shown to exhibit anti-tumor activity in vitro and in vivo. We investigated UCN-01 with respect to isozyme-specific PKC inhibition using purified recombinant or rabbit brain PKC isozymes, cPKC alpha, beta and gamma, nPKC delta, epsilon and eta, and a PKC zeta. Of the PKC isozymes examined, cPKC alpha was inhibited by UCN-01 most effectively (Ki = 0.44 nM), suggesting cPKC alpha is the prime candidate for the physiological target of UCN-01. The Ki values of UCN-01 estimated from Dixon plots for cPKC isozymes are approximately 1 nM, whereas the Ki values for nPKC isozymes are about 20 nM. Moreover, the Ki value for aPKC zeta is 3.8 microM. Thus, UCN-01 discriminates between PKC subfamilies. In addition, the inhibitory effects of staurosporine, H7, and calphostin C on aPKC zeta were examined and compared with those for cPKC alpha.

Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT), and alters gene expression in response to environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). We have previously shown that AhR contains both a nuclear localization signal (NLS), AhR(13-39), and a nuclear export signal (NES), AhR(55-75), in its NH(2)-terminal region. In this study, we obtained direct evidence for the nucleocytoplasmic shuttling of AhR and show the biological significance of the shuttling in terms of the transcriptional activation of its target gene, CYP1A1. When AhR(13-75) fused with glutathione S-transferase (GST)-green fluorescent protein (GFP) was microinjected into the nucleus of a polykaryotic of BHK21 cell, the GST-AhR(13-75)-GFP migrated from one nucleus to the other. This event, nucleocytoplasmic shuttling, was completely inhibited in the presence of leptomycin B (LMB). The interaction between chromosome region maintenance 1 (CRM1) and endogenous AhR was shown by immunoprecipitation with antibodies to AhR followed by immunoblot analysis with antibodies to CRM1. The inhibition of the nuclear export of AhR by LMB repressed the transcriptional activation of the CYP1A1 gene. The findings suggest that nuclear-cytoplasmic shuttling of AhR is essential for the inducible expression of the CYP1A1 protein.

Cellular and molecular effects of a pulse butyrate regimen and new inducers of globin gene expression and hematopoiesis.

Cooley's anemia is characterized by a deficiency of beta-globin chains, a relative excess of alpha-globin chains, and consequent accelerated programmed death of developing erythroid cells in the bone marrow. Increasing expression of the gamma-globin genes to adequately balance excess alpha-globin chains can ameliorate this disorder. Butyrates induce gamma-globin experimentally, but can also cause cell growth arrest with prolonged exposure or high concentrations, which in turn can accelerate apoptosis. To determine if these potentially opposing effects can be balanced to enhance therapeutic efficacy, an intermittent "pulsed" regimen of butyrate was evaluated. Following induction of gamma-globin mRNA and protein synthesis, total hemoglobin increased in beta-thalassemia patients by more than 2 g/dl above baseline, and Hb F increased above 20% in 5/8 sickle cell patients from baseline levels of 2% Hb F. Specific regulatory regions were identified in the gamma- and beta-globin gene promoters to which new binding of transcription factors, including alpha CP2 (an activator of gamma globin) occur during therapy solely in the butyrate-responsive patients. Other compounds which induce gamma globin, derivatives of acetic, phenoxyacetic, propionic, and cinnamic acids, and dimethylbutyrate, are under investigation. Some of these newer gamma-globin inducers (designed hemokines) provide better potential as therapeutics by also acting to increase hematopoietic cell viability and proliferation. Pharmacologic induction of expression of the endogenous gamma-globin genes is a realistic approach to therapy of the beta-globin disorders for many patients, with some effective agents available now and new therapeutics, with enhanced activities, under development.

Inhibition of proliferation and induction of differentiation of human myeloid leukemia cells by novel nucleoside analogs.

Purines such as hypoxanthine and 6-thioguanine have the capacity to induce the differentiation of human myeloid leukemia HL-60 cells in culture. Several nucleoside analogs were synthesized and their effects on cell proliferation and differentiation of HL-60 cells were examined. On incubation with these compounds, proliferation of HL-60 cells was inhibited and the cells were induced to differentiate into morphologically and functionally mature granulocytes. Among the compounds we tested, 2,4-diethyl-7,7,8,8-tetramethyl-cis-2,4-diazabicyclol [4.2.0] octane-3,5-dione was the most effective in inducing differentiation of HL-60 cells. This compound was approximately 100 times more potent on a molar basis than hypoxanthine. The compounds reacted synergistically or additively with a typical antileukemic drug (daunomycin) or another potent differentiation inducer (retinoic acid).