Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome.

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

Two distinct uricase II (nodulin 35) genes are differentially expressed in soybean plants.

Nodule-specific uricase (uricase II) is a homotetramer of a 33-kDa polypeptide, nodulin 35, and plays a key role in the assimilation of nitrogen fixed by microsymbionts in most legumes that have determinate nodules. We have isolated two distinct genes, UR2 and UR9, that encode for nodulin 35 from a soybean genomic library. Their corresponding cDNAs were also isolated from a nodule cDNA library. UR2 and UR9 both encode for 309 amino acid proteins with 12 amino acid differences. The expression of these two genes in various organs of soybean was examined by reverse transcription-polymerase chain reaction with primers specific to each cDNA sequences. Expression of UR9 was almost specific in root nodules, although it was expressed in roots, primary leaves, and developing seed at very low levels. In contrast, the UR2 transcripts were present in almost all plant organs at low levels, but no enhancement of the expression was observed in nodules. Thus, UR9 behaves as a nodulin gene, whereas UR2 is a nonsymbiotic uricase II gene. The sequences of their potential promoter regions share high homology within regions up to about 400 bp upstream from the translation initiation sites. These results suggest that symbiotic and nonsymbiotic uricase II genes diverged by gene duplication and that relatively small alterations in the promoter sequence enable the nodule-specific expression.

Structural and expression analysis of uricase mRNA from Lotus japonicus.

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.

Decreased levels of the androgen receptor in the mature rat phallus are associated with decreased levels of androgen receptor messenger ribonucleic acid.

Growth of the penis at sexual maturation is under the control of androgens, but growth ceases as the organ reaches adult size despite continued high levels of circulating androgen. Previous studies have shown that the cessation of penile growth is associated with a decrease in the quantity of androgen receptor, as detected by ligand binding and immunological methods. In the current studies we demonstrate that the decreased androgen receptor levels correlate with a decrease in androgen receptor mRNA content in the penile corpus and os, but not in the glans penis. These findings suggest that modulation of androgen receptor mRNA levels in the body of the penis may be important to the control of androgen-dependent growth in the tissue, and that the control of androgen receptor mRNA levels differs among the different cell types that comprise the penis. To explore the mechanisms controlling androgen receptor expression, we examined the transcription initiation site of the rat androgen receptor gene in ventral prostate and in three compartments of the penis: the corpus, the urethra, and the glans penis. The same promoter is employed in all preparations, suggesting that the different patterns of androgen receptor mRNA expression in these tissues with age are controlled by factors that modulate the activity of the same promoter.

Androgen receptor levels in the rat penis are controlled differently in distinctive cell types.

The rat phallus grows during sexual maturation as serum androgen concentrations rise to adult levels, and growth ceases when sexual maturity is attained despite the continued presence of adult serum androgen levels. This cessation of growth is correlated temporally with a diminution in the levels of androgen receptor, as detected by assays of ligand binding. To determine whether the change in androgen receptor content occurs in all cells in the tissue, immunohistochemical studies of the penile androgen receptor were performed in rats of different ages. In immature animals and animals castrated prepubertally, the androgen receptor is detected in virtually all cell types, including the corpus cavernosum penis, the small lateral cavernous bodies, the corpus cavernosum urethra, skin, urethra, and os penis. By contrast, in the mature rat penis minimal androgen receptor is evident within the corporal tissue and os, although immunoreactivity remains detectable in the penile skin and urethra. It is concluded that the cessation of androgen-mediated growth correlates with a decrease in androgen receptor levels in the body of the penis.

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Using beta-cell growth factors to enhance human pancreatic Islet transplantation.

This is a particularly exciting time in the field of pancreatic islet growth, development, and survival. The recent publication of a study demonstrating that human pancreatic islet transplantation is both technically and immunologically feasible has highlighted the need for large supplies of pancreatic islets or pancreatic beta cells for larger-scale islet transplantation in patients with diabetes. This, together with a rapid expansion in the past several years of the understanding of mechanisms of islet growth, development, and survival, has accelerated and invigorated efforts to therapeutically harness the cellular mechanisms responsible for pancreatic beta-cell proliferation, survival, and development and to take advantage of this new knowledge to enhance the availability, survival, and function of pancreatic beta cells in human islet transplantation for diabetes mellitus. Here, we briefly review the confluence of events that have provided optimism and energy to the islet transplant field, and we focus on peptide growth factors that eventually may be deployed in the effort to augment islet mass and function in patients with diabetes.

Parathyroid hormone-related protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets.

The cloning of the so-called 'parathyroid hormone-related protein' (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N-terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly-hormone which comprises a family of distinct peptide hormones arising from post-translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N-terminal, mid-region and C-terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1-34) and PTHrP(1-36), is the only cloned receptor so far. PTHrP is a PTH-like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo-vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium-sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

Functional analysis of the human androgen receptor promoter.

Although androgen receptor levels vary widely during development, our previous studies have suggested that a single promoter is active in a number of human and rat tissues and cell lines. To examine the elements controlling androgen receptor expression, we performed deletion mapping and site-directed mutagenesis of the human androgen receptor promoter and assayed these promoter fusions in human cell lines that produce the androgen receptor as well as those deficient in the androgen receptor, both in the presence and absence of androgen. Our studies identify a region (-74 to +87) surrounding the site of transcription initiation that constitutes the core of the androgen receptor promoter. When assayed in T47D cells (an AR-expressing cell line), this segment was sufficient to direct the expression of reporter genes at levels similar to that observed for larger promoter fragments, and further deletions led to an appreciable loss of promoter activity. DNA sequences surrounding this region appear to modulate the activity of this minimal promoter in a cooperative fashion. Site-directed mutagenesis and mobility shift assays demonstrated the importance of regulatory regions upstream of the transcription initiation site including an SP1 binding site and two segments with similarities to consensus HLH protein binding sites (E-boxes). Androgen treatment did not cause a decrease in activity of any of the transiently transfected promoter fusions tested, suggesting that the promoter does not contain the information necessary for autoregulation or that a posttranscriptional or posttranslational mechanism is responsible for the regulation of AR mRNA by ligand. Furthermore, the androgen receptor promoter fusions displayed differing transcriptional activities when transfected into two cell lines deficient in androgen receptor expression suggesting heterogeneity in the mechanisms responsible for this deficiency.

Ph1-positive CML-derived myeloid-monocytoid precursor cell line producing substance(s) that stimulates normal CFU-C.

A new Ph1-chromosome positive cell line, KOPM-28. was established from a patient with chronic myelogenous leukemia (CML) in blast crisis. KOPM-28 cells were phenotypically immature: without azurophilic granules; negative for myeloperoxidase and positive for specific and nonspecific esterases. The nonspecific esterase reaction was intensified by TPA, and retinoic acid reinforced the specific esterase reaction without inducing morphological changes. KOPM-28 cells were not phagocytic. The cells expressed complement receptors, myeloid-monocytoid antigens, an Ia-like antigen and T4 antigen. CALLA, T-lymphocyte specific antigens, B-lymphocyte related antigen and platelet-megakaryocyte-megakaryoblast specific antigen were not detected. KOPM-28 cells formed colonies in semi-solid medium; this ability was augmented by GM-CSA. The addition of culture medium conditioned by KOPM-28 cells to normal bone marrow cells resulted in the increase of the CFU-C colonies. These findings indicate that KOPM-28 cells have features of myeloid and monocytoid precursor cells and that they are producing substance(s) which stimulates normal CFU-C.

Antimalarial activity of extracts of Malaysian medicinal plants.

In vitro and in vivo studies revealed that Malaysian medicinal plants, Piper sarmentosum, Andrographis paniculata and Tinospora crispa produced considerable antimalarial effects. Chloroform extract in vitro did show better effect than the methanol extract. The chloroform extract showed complete parasite growth inhibition as low as 0.05 mg/ml drug dose within 24 h incubation period (Andrographis paniculata) as compared to methanol extract of drug dose of 2.5 mg/ml but under incubation time of 48 h of the same plant spesies. In vivo activity of Andrographis paniculata also demonstrated higher antimalarial effect than other two plant species.

[Monocytic crisis in chronic myeloid leukemia: a case report].

We report a 17-year-old female with chronic myeloid leukemia (CML) who developed monocytic crisis. She was diagnosed as chronic phase of Ph1-chromosome positive CML at 14 years old. Three years after the diagnosis of the disease, she was admitted to the hospital because of low grade fever, lethargy and marked splenomegaly. Small dose of Ara-C relieved her symptoms and splenomegaly. Six months later, however, a marked leukocytosis over 70,000/microliters were observed, and the peripheral blood smear disclosed that about 80% of the leukocytes were relatively mature monocytoid cells. Chromosomal analysis revealed additional abnormalities (double Ph1, +8, +9, +19). Lysozyme levels in serum and urine were high and NAP score was elevated. These monocytoid cells expressed receptors for IgG-Fc and C3, phagocytic activity, and monocytoid antigens which were determined by monoclonal antibodies (MY4, Mo2, OKM5). Cytochemically, almost all of monocytoid cells were positive for peroxidase and naphthol-ASD-chloroacetate esterase (CAE), but the monocytoid cells positive for non-specific esterase were limited. These data suggested that this case was monocytic crisis in CML with proliferation of CAE positive monocytoid cells. Among several types of blast crisis, monocytic crisis is extremely rare condition. The definite monocytic crisis demonstrated by this case may support the hypothesis that target cells of CML are pluripotent hematopoietic precursors.

Androgen receptor of rat penis is down-regulated by androgen.

To provide insight into the factors that control androgen receptor levels in rat penis, we assessed 5 alpha-[3H]-dihydrotestosterone binding in low-salt [10 mM tris(hydroxymethyl)aminomethane (Tris), 10 mM Na2M0O4] and high-salt (10 mM Tris, 10 mM Na2M0O4, 0.5 M KCl) extracts of rat penis using sucrose density gradients. Total receptor content decreased from approximately 729 +/- 114 fmol/g tissue at 3 wk of age to less than 50 fmol/g tissue at 10 wk of age. Castration of 3-wk-old rats prevented penile growth and the age-related decline in penile androgen receptor. Treatment of 3-wk-old castrated rats with 5 alpha-dihydrotestosterone caused an acceleration in the decline in receptor levels compared with intact animals. Castration of 10-wk-old rats (after androgen receptor levels had decreased) did not result in an increase in the amount of total androgen receptor by 16 wk of age. To determine the specificity of the androgen-mediated decline in receptor levels, the amounts of prostate androgen receptor were compared with those of the penis at different ages. When expressed as femtomoles per organ, the total androgen receptor level in the prostate increased fourfold from 3 to 10 wk of age, whereas the total androgen receptor in the penis declined approximately threefold. We conclude that the downregulation of the penile androgen receptor content that occurs in the rat between 3 and 10 wk of age is androgen mediated, does not occur in all androgen target tissues, and is prevented but not reversed by castration.

Expression of two uricase (Nodulin-35 genes in a non-ureide type legume, Medicago sativa.

Two cDNA clones, MsU2 and MsU9 encoding uricase (EC 1.7.3.3, Nodulin-35) were isolated from a cDNA library prepared from nodule tissues of alfalfa, Medicago sativa, plants. Both MsU2 and MsU9 encoded 308 amino acid polypeptides with a difference of 5 amino acids, and the deduced amino acid sequences shared 98% homology. Between these two cDNA clones and uricase genes of soybean which were designated as Nod-35s, more than 80% identity was observed in nucleotides and deduced amino acid sequences, suggesting that these MsU2 and MsU9 are homologs of Nod-35. Using the reverse transcription-PCR technique, we detected the transcripts of these two genes in almost all tissues of alfalfa. The operation of uricase genes was confirmed by the presence of ureide in the xylem sap and uricase activity in the nodules. In situ hybridization analysis revealed that MsU2 and MsU9 were expressed only in uninfected cells of the infected zone of the nodule tissue. The cell specific-expression of the two uricase genes was observed in an identical manner to that of Nod-35 in soybean nodules.