Continuous MLL-ENL expression is necessary to establish a "Hox Code" and maintain immortalization of hematopoietic progenitor cells

The t[(11;19)(p22;q23)] translocation, which gives rise to the MLL-ENL fusion protein, is commonly found in infant acute leukemias of both the myeloid and lymphoid lineage. To investigate the molecular mechanism of immortalization by MLL-ENL we established a Tet-regulatable system of MLL-ENL expression in primary hematopoietic progenitor cells. Immortalized myeloid cell lines were generated, which are dependent on continued MLL-ENL expression for their survival and proliferation. These cells either terminally differentiate or die when MLL-ENL expression is turned off with doxycycline. The expression profile of all 39 murine Hox genes was analyzed in these cells by real-time quantitative PCR. This analysis showed that loss of MLL-ENL was accompanied by a reduction in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte colony-stimulating factor, we show for the first time that reduced Hox gene expression is specific to loss of MLL-ENL and is not a consequence of differentiation. Our data also suggest that the Hox cofactor Meis-2 can substitute for Meis-1 function. Thus, MLL-ENL is required to initiate and maintain immortalization of myeloid progenitors and may contribute to leukemogenesis by aberrantly sustaining the expression of a "Hox code" consisting of Hoxa4 to Hoxa11.

A novel base change leading to Hb vanderbilt [β89(F5)Ser→Arg, AGT>AGA]

We describe a high oxygen affinity hemoglobin (Hb) variant (Hb Vanderbilt) as a result of a heterozygous novel base change from T to A at codon 89 (AGT>AGA) leading to an amino acid change from serine to arginine. © 2011 Informa Healthcare USA, Inc.

Low-dose salinomycin induces anti-leukemic responses in AML and MLL

Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response.
Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.

Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum

The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) M. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides.

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT-PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

Altered expression of the septin gene, SEPT9, in ovarian neoplasia.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non-malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5-azacytidine treatment, suggesting a role for methylation in the control of expression of this gene. Copyright © 2003 John Wiley & Sons, Ltd.

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

We have isolated a novel bradykinin B2-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.

No association between hOGG1, XRCC1, and XPD polymorphisms and risk of reflux esophagitis, Barrett's Esophagus, or esophageal adenocarcinoma: Results from the factors influencing the Barrett's adenocarcinoma relationship case-control study

Reflux of gastric contents can lead to development of reflux esophagitis and Barrett's esophagus. Barrett's esophagus is a risk factor for esophageal adenocarcinoma. Damage to DNA may lead to carcinogenesis but is repaired through activation of pathways involving polymorphic enzymes, including human 8-oxoguanine glycosylase 1 (hOGG1), X-ray repair cross-complementing 1 (XRCC1), and xeroderma pigmentosum group D (XPD). Of the single nucleotide polymorphisms identified in these genes, hOGG1 Ser 326Cys, XRCC1 Arg 399Gln, and XPD Lys 751Gln are particularly common in Caucasians and have been associated with lower DNA repair capacity. Small studies have reported associations with XPD Lys 751Gln and esophageal adenocarcinoma. XRCC1 Arg 399Gln has been linked to Barrett's esophagus and reflux esophagitis. In a population-based case-control study, we examined associations of the hOGG1 Ser 326Cys, XRCC1 Arg 399Gln, and XPD Lys 751Gln polymorphisms with risk of esophageal adenocarcinoma, Barrett's esophagus, and reflux esophagitis. Genomic DNA was extracted from blood samples collected from cases of esophageal adenocarcinoma (n = 210), Barrett's esophagus (n = 212), reflux esophagitis (n = 230), and normal population controls frequency matched for age and sex (n = 248). Polymorphisms were genotyped using Taq-Man allelic discrimination assays. Odds ratios and 95% confidence intervals were obtained from logistic regression models adjusted for potential confounding factors. There were no statistically significant associations between these polymorphisms and risk of esophageal adenocarcinoma, Barrett's esophagus, or reflux esophagitis.

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine 0-sulfation motif.

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

Helokinestatin: a new bradykinin B2 receptor antagonist decapeptide from lizard venom.

Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Mr 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.

Advanced glycation of fibronectin impairs vascular repair by endothelial progenitor cells: Implications for vasodegeneration in diabetic retinopathy

PURPOSE. Vascular repair by marrow-derived endothelial progenitor cells (EPCs) is impaired during diabetes, although the precise mechanism of this dysfunction remains unknown. The hypothesis for the study was that progressive basement membrane (BM) modification by advanced glycation end products (AGEs) contributes to impairment of EPC reparative function after diabetes-related endothelial injury.

METHODS. EPCs isolated from peripheral blood were characterized by immunocytochemistry and flow cytometry. EPC interactions on native or AGE-modified fibronectin (AGE-FN) were studied for attachment and spreading, whereas chemotaxis to SDF-1 was assessed with the Dunn chamber assay. In addition, photoreactive agent-treated monolayers of retinal microvascular endothelial cells (RMECs) produced circumscribed areas of apoptosis and the ability of EPCs to “endothelialize” these wounds was evaluated.

RESULTS. EPC attachment and spreading on AGE-FN was reduced compared with control cells (P < 0.05–0.01) but was significantly restored by pretreatment with Arg-Gly-Asp (RGD). Chemotaxis of EPCs was abolished on AGE-FN but was reversed by treatment with exogenous RGD. On wounded RMEC monolayers, EPCs showed clustering at the wound site, compared with untreated regions (P < 0.001); AGE-FN significantly reduced this targeting response (P < 0.05). RGD supplementation enhanced EPC incorporation in the monolayer, as determined by EPC participation in tight junction formation and restoration of transendothelial electric resistance (TEER).

CONCLUSIONS. AGE-modification of vascular substrates impairs EPC adhesion, spreading, and migration; and alteration of the RGD integrin recognition motif plays a key role in these responses. The presence of AGE adducts on BM compromises repair by EPC with implications for vasodegeneration during diabetic microvasculopathy.