Overproduction Of Cells Research Articles

The role of her4 in inner ear development and its relationship with proneural genes and Notch signalling.

The generation of sensory neurons and hair cells of the inner ear is under tight control. Different members of the Hairy and Enhancer of Split genes (HES) are expressed in the inner ear, their full array of functions still not being disclosed. We have previously shown that zebrafish her9 acts as a patterning gene to restrict otic neurogenesis to an anterior domain. Here, we disclose the role of another her gene, her4, a zebrafish ortholog of Hes5 that is expressed in the neurogenic and sensory domains of the inner ear. The expression of her4 is highly dynamic and spatiotemporally regulated. We demonstrate by loss of function experiments that in the neurogenic domain her4 expression is under the regulation of neurogenin1 (neurog1) and the Notch pathway. Moreover, her4 participates in lateral inhibition during otic neurogenesis since her4 knockdown results in overproduction of the number of neurog1 and deltaB-positive otic neurons. In contrast, during sensorigenesis her4 is initially Notch-independent and induced by atoh1b in a broad prosensory domain. At later stages her4 expression becomes Notch-dependent in the future sensory domains but loss of her4 does not result in hair cell overproduction, suggesting that there other her genes can compensate its function.

Friedreich's Ataxia: A Neuronal Point of View on the Oxidative Stress Hypothesis.

A prominent feature of Friedreich’s ataxia (FRDA) is the neurodegeneration of the central and peripheral nervous systems, but little information is available about the mechanisms leading to neuronal damage in this pathology. Currently, no treatments delay, prevent, or reverse the inexorable decline that occurs in this condition. Evidence of oxidative damage has been demonstrated in Friedreich’s ataxia, and this damage has been proposed as the origin of the disease. Nevertheless, the role of oxidative stress in FRDA remains debatable. The lack of direct evidence of reactive oxygen species overproduction in FRDA cells and tissues and the failure of exogenous antioxidants to rescue FRDA phenotypes questions the role of oxidative stress in this pathology. For example, the antioxidant “idebenone” ameliorates cardiomyopathy in FRDA patients, but this therapy does not improve neurodegeneration. To date, no known pharmacological treatment with antioxidant properties cures or delays FRDA neuropathology. This review reports and discusses the evidence of oxidative stress in FRDA and focuses on the existing knowledge of the apparent ineffectiveness of antioxidants for the treatment of neuronal damage.

Epidemiology of MPN: what do we know?

The myeloproliferative neoplasms, are characterised by overproduction of myeloid cells. Chronic myeloid leukaemia, polycythaemia vera, essential thrombocythaemia, myelofibrosis and the very rare disorders chronic neutrophilic leukaemia, chronic eosinophilic leukaemia not otherwise specified and mastocytosis are all included in the group. Incidence and prevalence rates reported in the worldwide literature are presented in this review. Survival data on each condition is described. Information on the aetiology of the disorders is discussed including body mass index, diet, smoking and alcohol, allergies, associated medical conditions, occupation and environmental exposures with focus on recent new studies. The aetiology of the myeloproliferative neoplasms remains unknown, and this review of the current state of knowledge highlights the need for further comprehensive research.

The therapeutic potential of cystathionine β-synthetase/hydrogen sulfide inhibition in cancer.

Cancer represents a major socioeconomic problem; there is a significant need for novel therapeutic approaches targeting tumor-specific pathways. In colorectal and ovarian cancers, an increase in the intratumor production of hydrogen sulfide (H2S) from cystathionine β-synthase (CBS) plays an important role in promoting the cellular bioenergetics, proliferation, and migration of cancer cells. It also stimulates peritumor angiogenesis inhibition or genetic silencing of CBS exerts antitumor effects both in vitro and in vivo, and potentiates the antitumor efficacy of anticancer therapeutics. Recently published studies are reviewed, implicating CBS overexpression and H2S overproduction in tumor cells as a tumor-growth promoting "bioenergetic fuel" and "survival factor," followed by an overview of the experimental evidence demonstrating the anticancer effect of CBS inhibition. Next, the current state of the art of pharmacological CBS inhibitors is reviewed, with special reference to the complex pharmacological actions of aminooxyacetic acid. Finally, new experimental evidence is presented to reconcile a controversy in the literature regarding the effects of H2S donor on cancer cell proliferation and survival. From a basic science standpoint, future directions in the field include the delineation of the molecular mechanism of CBS up-regulation of cancer cells and the delineation of the interactions of H2S with other intracellular pathways of cancer cell metabolism and proliferation. From the translational science standpoint, future directions include the translation of the recently emerging roles of H2S in cancer into human diagnostic and therapeutic approaches.

Endoplasmic reticulum stress promotes amyloid-beta peptides production in RGC-5 cells

Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including Alzheimer's disease. We have previously observed amyloid production in the retina of the Tg2576 transgenic mouse model of Alzheimer's disease. In this study, we used tunicamycin-induced ER stress in RGC-5 cells, a cell line identical to the photoreceptor cell line 661W, to investigate the effect of ER stress on production of amyloid-beta (Abeta) peptides. We found that the mRNA level of amyloid-beta precursor protein (APP) remained stable, while the protein level of amyloid-beta precursor protein (APP) was decreased, the amyloid-beta precursor protein cleaving enzymes beta-site APP-cleaving enzyme 1 and presenilin 1 were upregulated, Abeta1-40 and Abeta1-42 production were increased, and reactive oxygen species production and apoptosis markers were elevated following induction of ER stress. The protein level of Abeta degradation enzymes, neprilysin, endothelin-converting enzyme 1, and endothelin-converting enzyme 2 remained unchanged during the prolonged ER stress, showing that the generation of Abeta did not result from reduction of proteolysis by these enzymes. Inclusion of group II caspase inhibitor, Z-DEVD-FMK, increased the ER stress mediated Abeta production, suggesting that they are generated by a caspase-independent mechanism. Our findings provided evidence of a role of ER stress in Abeta peptide overproduction and apoptotic pathway activation in RGC-5 cells.

Glycyrrhizin inhibits human neutrophil elastase-induced mucin 5AC overproduction in human bronchial epithelial cells

To investigate the effect of glycyrrhizin (Gly) on human neutrophil elastase (HNE)- induced mucin (MUC) 5AC overproduction in human bronchial epithelial cells (16HBE), and the potential signaling pathway involved in this process. The cultured cells were divided into 3 groups: a control group, cultured in serum-free DMEM medium; an HNE group, pretreated with HNE alone; and a Gly group, incubated with HNE and Gly. After stimulation with a variety of Gly concentrations, the cytotoxicity was assessed by methyl thiazolyl tetrazolium method. The mRNA expressions of p38, nuclear factor κB (NF-κB) p65, inhibitory κBα (IκBα) and MUC5AC were detected by real-time PCR. The phosphorylation levels of p38 (p-p38), NF-κB p65 (p-NF-κB p65) and IκBα (p-IκBα) were measured by Western blot while the levels of MUC5AC protein were analyzed by emzyme-linked immunosorbent assay and immunofluorescence. Compared with the control group, the expression levels of MUC5AC mRNA and protein in the HNE group were both significantly increased. There was a significant increase in p-p38 and p-NF-κB p65, while the production of IκBα was much lower than that in the control group. Gly significantly inhibited the increase of MUC5AC, p38 and NF-κB p65, but increased the activity of IκBα. Glycyrrhizin can inhibit MUC5AC overproduction via p38-NF-κB p65/IκBα signaling pathway.

Autoimmune Disease pH and Temperature.

Perhaps parts of the immune system are pH and temperature sensitive. If true, there could be a variance in pH and/or temperature under certain conditions that could impact the immune system and vice versa. Possibly the illusive etiology of autoimmune disease is in part due to pH and/or temperature changes. A majority would probably agree the human immune system is designed to destroy foreign invasion such as an infection and/or for removal of nonviable tissue such as that damaged by trauma. Obviously, both of the previous situations would be more likely to involve at least a local environment trending more acidic. Also most would agree autoimmune disease is when there is an error in the immune system and the system begins to attack and destroy viable tissue. Thus could the etiology of autoimmune disease be a repair system gone awry possibly because of a pH and/ or temperature change? If cancer is the repair system gone awry in regard to overproduction of replacement cells, then perhaps autoimmune disease is the repair system gone awry in regard to the damaged cell removal causing disease possibly because of a pH and/or temperature change. When there is cell damage inflammation develops as certain cells infiltrate the area and certain mediators accumulate that are also part of the immune system. Autoantibodies have been noted to be present following trauma with some suspecting this is a mechanism to remove cellular debris. However, autoantibody presence suggests a possible autoimmune disease in the future but is not diagnostic, since many normal individuals have mild levels. For example, with diabetes mellitus type I, there can be an autoantibody present for years (or even a life time) without any overt disease ever developing. In short, autoimmunity may follow tissue damage with low levels of antibodies but not necessarily actual autoimmune disease with destruction of viable tissue. So this is similar to other situations where something could occur, but does not have to occur, or rather something is more likely to occur, but is still not a certainty. An unknown second step probably has to occur, could that second step involve a pH and/or temperature change is the question. If an individual develops an autoimmune disease, they are more likely to develop a second autoimmune disease than are other patients. Autoimmune diseases seem to run in families as well. There appears to be a genetic susceptibility. Some say autoimmune diseases are multigenic, heterogeneous in genetic basis, with identical twins mixed on penetrance but usually higher than siblings and much higher than unrelated populations. If there is a pH aspect to the immune system, perhaps this involves the cellular pumps, channels, transporters and isoenzyme levels. This could account for a familial predisposition yet be multigenic, since there are so many different cellular pumps, channels, transporters and isoenzymes. There are multiples of each of these, so a particular combination could make an individual susceptible and this would be genetic. The cellular pumps, channels, transporters and isoenzymes could also respond to environmental stress changing to some degree accounting for differences in identical twins. The medications procainamide and hydralazine have been known for decades to induce autoimmune disease, specifically lupus. Procainamide impacts sodium channels while hydralazine impacts the calcium activated potassium channels and so both could impact pH levels. Interestingly, some compliment activation [1] and IgG binding [2] are more reactive in an acidic environment. In addition, some interleukins seem to prefer an acidic environment [3]. But there is also a possibility extreme levels of alkalinity can set the immune system into action as well, at the very least through cell destruction. Strong ions such as iodine in the thyroid could conceivably impact the cellular pumps, channels, transporters and isoenzymes. Indeed that is possibly what we see. As the level of intrathyroidal iodine increases, so does the incident of Hashimoto’s thyroiditis [4], one of the most common autoimmune diseases. However, not everyone develops Hashimoto’s thyroiditis with higher iodine, just some people. Could those with a certain mix of cellular pumps, channels, transporters and isoenzymes be the ones that are more likely to develop the Hashimoto’s thyroiditis? Also some cytokines such as interferon can induce Hashimoto’s thyroiditis. Could interferon possibly be impacting the cellular pumps, channels, transporters and isoenzymes? Yes, it is known to impact the potassium channels for example [5]. Finally, if pH changes enough there could be a charge generated which could impact movement possibly even for T and B cells. Another interesting aspect about autoimmune diseases is they are in general significantly more common usually in females than males. Women have increased antibody production compared to Manuscript accepted for publication April 10, 2014

Aging and impaired hematopoiesis

기능이상과 같은 것이다. 이와 반대로 마모(wear and tear) Aging is an universal phenomenon and irreversible syndrome, and its damage occurs to molecules (DNA, proteins, and lipids), to cells, and to organs. Hematopoietic tissue intrinsically has a very high turnover rate; nonetheless, it is not protected from age-related insults. Aging results in the overproduction of myeloid cells, which leads to a pro-inflammatory environment. The selective expansion of a clonal subtype of intrinsically myeloid-biased hematopoietic stem cells (HSCs) is a central component of hematopoietic aging. In the present study, the stress-response and inflammatory genes were up-regulated with age whereas chromatin remodeling and DNA repair genes were down-regulated. Accumulated DNA damage, loss of DNA repair, and epigenetic deregulation are the main molecular mechanisms underlying age-dependent HSC decline. The most profound effect is seen in the adaptive immune system with a marked decline of lymphoid function in the elderly. Mitochondrial dysfunction and mitochondrial DNA mutation are another important contributor to the aging of HSCs, which have been regarded as a part of the mitochondrial theory of aging. Generation of reactive oxygen species during mitochondrial adenosine triphosphate generation, results in damage to mitochondria and mitochondrial DNA, the latter leading to deleterious mutations that directly caused the functional decline of human. Studies have pointed toward intrinsic deficits in HSC function, and epigenetic deregulation as the important contributing factors behind hematopoietic decline and malignancy during aging. Aging-related changes such as hematopoiesis are reflected by a decline in marrow cellularity, increased risk of anemia, marrow failure syndrome, and myeloproliferative neoplasms as well as a decline of adaptive immunity.

The Genomic Landscape of Myeloproliferative Neoplasms: Somatic Calr Mutations in the Majority of JAK2-Wildtype Patients

BCR-ABL negative myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are chronic myeloid malignancies characterized by overproduction of hematopoietic cells. JAK2 mutations are found in most patients with PV, and in only 50-60% of patients with ET and MF. JAK2 mutation testing has greatly simplified MPN diagnosis, but distinguishing JAK2-wildtype ET from reactive thrombocytosis remains a diagnostic challenge. Mutations in signalling pathways (MPL, LNK) and epigenetic regulators (TET2, DNMT3A, IDH1/2, EXH2, ASXL1) have been found in a minority of MPNs. However genome-wide data are lacking and the pathogenesis of MPNs that do not harbor JAK2 or MPLmutations remains obscure. MethodsExome sequencing was performed in 151 MPN patients on matched tumor and constitutional samples. CALR status was assessed in 3412 samples using Sanger sequencing and analysis of exome/genome sequencing data. Presence of CALR mutations in hematopoietic stem and progenitor cells was assessed by flow sorting and sequencing. Phylogenetic trees were established using hematopoietic colonies. Calreticulin cellular localisation was assessed in patient samples and cell lines expressing CALR variants by flow cytometry and immunofluorescence. ResultsExome sequencing identified 1498 somatic mutations with a median of 6.5 mutations in PV and ET, and 13 in MF (MF vs ET, P=0.0002; MF vs PV, P=0.008). JAK2V617F was found in all cases of PV (n=48), 56% of ET (35/62), and 69% of MF (27/39), and MPL mutations in 7 ET and MF cases. Mutations in epigenetic regulators TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 were identified in 22, 12, 12, 4, 3 patients respectively, and components of the splicing machinery (U2AF1, SF3B1 or SRSF2) were mutated in 9 patients. Mutations in rare genes reported to be mutated in MPNs were found in four patients (1 CBL; 2 NFE2; 1 SH2B3/LNK). We found novel somatic mutations in CHEK2 (1 PV, 1 ET and 1 MF) which have not been previously reported in MPNs. The mutation spectrum showed a predominance of C>T transitions. Pairwise associations between MPN genes demonstrated that ASXL1 and SRSF2 mutations were positively correlated with mutations in epigenetic modifiers.Novel somatic mutations in calreticulin (CALR) were identified by exome sequencing in the majority (26/31) of JAK2 or MPL unmutated patients. CALR and JAK2/MPL mutations were mutually exclusive, and 97% of patients harbored a mutation in 1 of these 3 genes. In an extended follow up screen of 1345 hematological malignancies, 1517 other cancers and 550 controls we found CALR mutations in 71% of ET (80/112), 56% of idiopathic MF (18/32), 86% of post ET-MF (12/14) and 8% of myelodysplasia (10/115), but not in other myeloid, lymphoid or solid cancers. Compared to JAK2-mutated MPNs, those with CALR mutations presented with higher platelet counts (Wilcoxon rank-sum, P=0.0003), lower hemoglobin levels (Student's t test, P=0.02) and showed a higher incidence of transformation to MF (Fishers exact, P=0.03).All CALR mutations were insertions or deletions affecting exon 9, with 2 common variants L367fs*46 (52 bp deletion) and K385fs*47 (5 bp insertion). Loss of heterozygosity over CALR was seen in a minority of patients. Of 148 CALR mutations identified, there were 19 distinct variants. Remarkably, all generated a +1 basepair frameshift, which results in loss of most of the C-terminal acidic domain of the protein as well as the KDEL Golgi-to-endoplasmic reticulum (ER) retrieval signal, raising the possibility of compromised ER retention. Mutant proteins were readily detected in transfected cell lines and localised to the ER in the same manner as wildtype CALR, without Golgi or cell surface accumulation. These results are consistent with studies reporting KDEL-independent mechanisms of ER retention.Mutation of CALR was detected in highly purified hematopoietic stem/progenitor cells. Clonal analyses demonstrated CALR mutations in the earliest phylogenetic node in 5/5 patients, consistent with it being an initiating mutation in these individuals. ConclusionsWe describe the mutational landscape of BCR-ABL negative MPNs and demonstrate that somatic mutations in the endoplasmic reticulum chaperone CALR are found in the majority of patients with JAK2-unmutated MPNs. These results reveal a novel biological pathway as a target for tumorigenic mutations and will simplify diagnosis of MPN patients. Disclosures:Bowen:Celgene: Honoraria. Harrison:S Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Phosphatidylinositol-3-Kinase p110d Uniquely Promotes Gain-Of-Function PTPN11-Induced GM-CSF Hypersensitivity In a Model Of Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a fatal leukemia affecting children under the age of 4 years and is characterized by myelomonocytic cell overproduction and hypersensitivity to GM-CSF. The only curative therapy is allogeneic stem cell transplantation; however, half of children relapse after this aggressive therapy. Approximately 85% of JMML patients bear loss-of-function (LOF) mutations in NF1 or CBL or gain-of-function (GOF) mutations in KRAS, NRAS, or PTPN11. Typically, these mutations are non-overlapping, with the net effect being Ras hyperactivation. Children bearing somatic GOF mutations within PTPN11, which encodes the protein tyrosine phosphatase, Shp2, exhibit the poorest prognosis. GOF Shp2 (Shp2D61Y and Shp2E76K) induces hyperactivation of both the Ras-MEK- Erk and PI3K-Akt pathways. While the Ras-MEK-Erk pathway clearly contributes to the pathogenesis of JMML, we hypothesize that the PI3K-Akt pathway cooperates with the Ras-MEK-Erk pathway to promote JMML. Recently published work indicates that genetic disruption of the PI3K regulatory subunit, p85a, reduces GOF Shp2-induced hypersensitivity to GM-CSF. However, as PI3K regulatory subunits cannot be easily inhibited pharmacologically, we examined the contribution of class IA PI3K catalytic subunits in GOF Shp2-induced JMML.Shp2D61Y/+;Mx1Cre+ mice were crossed with mice bearing conditional knockout of p110a (Pik3caflox/flox) or bearing a kinase dead mutant of p110d (Pik3cdD910A/D910A). Shp2D61Y/+;Mx1Cre-, Shp2D61Y/+;Mx1Cre+, Shp2D61Y/+;Mx1Cre+; Pik3caflox/flox, and Shp2D61Y/+;Mx1Cre+; Pik3cdD910A/D910A mice were treated with polyI;polyC, and 8 weeks post-treatment, animals were euthanized followed by evaluation of spleen size, hypersensitivity of bone marrow low density mononuclear cells (LDMNCs) to GM-CSF, frequency of bone marrow phenotypically-defined common myeloid, granulocyte-monocyte, and megakaryocyte-erythroid progenitors (CMPs, GMPs, and MEPs), and GM-CSF-stimulated Erk and Akt activation. Genetic disruption of p110a failed to normalize GOF Shp2-induced splenomegaly, GM-CSF hypersensitivity in proliferation assays and methylcellulose-based progenitor assays, or hyperphosphorylation of Erk or Akt. In contrast, genetic ablation of p110d kinase activity significantly reduced spleen size, normalized progenitor hypersensitivity to GM-CSF, and reduced both Akt and Erk hyperactivation. Additionally, genetic inhibition of p110d normalized the skewed hematopoietic progenitor distribution reported in the Shp2D61Y/+;Mx1Cre+ mice, while genetic disruption of p110a failed to do so. This unique function of p110d in the context of GOF Shp2-expressing mice is significant, as p110d expression is restricted to hematopoietic cells and p110d bears transforming properties independent of Ras.While previously published work indicates that the PI3K p110a and p110d inhibitor, GDC-0941, inhibits proliferation of GOF Shp2-expressing cells, we tested if the potent p110d-specific inhibitor, GS-9820, is similarly effective. GOF Shp2-expressing bone marrow LDMNCs treated with GS-9820 demonstrated significantly reduced proliferation in a dose-dependent fashion, while GS-9820 failed to inhibit the proliferation of WT Shp2-expressing cells. GS-9820 treatment decreased Akt phosphorylation (S473 and T308) as well as reduced Erk phosphorylation, indicating that p110d inhibition also reduces signaling within the Ras-MEK-Erk pathway. While PI3K activates the canonical Akt-mTORC1 pathway, it also positively feeds back to the Ras-MEK-Erk pathway via activation of Rac-Pak-MEK; therefore, we evaluated if p110d inhibition adds to or is redundant with MEK inhibition. Treatment of GOF Shp2-expressing hematopoietic cells with the MEK inhibitor, PD0325901, effectively reduced proliferation, and addition of GS-9820 further significantly reduced proliferation, indicating that p110d works cooperatively with MEK to promote GOF Shp2-induced disease.Collectively, our findings suggest that PI3K catalytic subunit p110d functions in a Ras-MEK-Erk pathway-independent manner to promote GOF Shp2-induced hypersensitivity to GM-CSF, and suggest that PI3K p110d inhibition in combination with MEK inhibition may be a novel, optimal approach for the treatment of JMML. Disclosures:No relevant conflicts of interest to declare.

The Diadenosine Homodinucleotide P18 Improves In Vitro Myelination in Experimental Charcot‐Marie‐Tooth Type 1A

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca(2+) concentration ([Ca(2+)]i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca(2+) levels through down-regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5',5'''-P1, P2-diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11-overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild-type (wt) cells. P18 increased [cAMP]i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non-phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]i-increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype.

Procollagen I-expressing renin cell precursors

Renin-expressing cells in the kidney normally appear as mural cells of developing preglomerular vessels and finally impose as granulated juxtaglomerular cells in adult kidneys. The differentiation of renin-expressing cells from the metanephric mesenchyme in general and the potential role of special precursor stages in particular is not well understood. Therefore, it was the aim of this study to search for renin cell precursors in the kidney. As an experimental model, we used kidneys of aldosterone synthase-deficient mice, which display a prominent compensatory overproduction of renin cells that are arranged in multilayered perivascular cell clusters. We found that the perivascular cell clusters contained two apparently distinct cell types, one staining positive for renin and another one staining positive for type I procollagen (PC1). It appeared as if PC1 and renin expression were inversely related at the cellular level. The proportion of renin-positive to PC1-positive cells in the clusters was inversely linked to the rate of salt intake, as was overall renin expression. Our findings suggest that the cells in the perivascular cell clusters can reversibly switch between PC1 and renin expression and that PC1-expressing cells might be precursors of renin cells. A few of those PC1-positive cells were found also in adult wild-type kidneys in the juxtaglomerular lacis cell area, in which renin expression can be induced on demand.

Inhibition of SRC Corrects GM-CSF Hypersensitivity That Underlies Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm in children characterized by the overproduction of monocytic cells that infiltrate the spleen, lung, and liver. JMML remains a disease for which few curative therapies are available other than myeloablative hematopoietic stem cell transplant (HSCT); however, relapse remains a major cause of treatment failure and the long-term morbidities of HSCT for survivors are substantial. A hallmark feature of JMML is acquired hypersensitivity by clonal myeloid progenitor cells to granulocyte macrophage-colony stimulating factor (GM-CSF) via a largely unknown mechanism. Here, we identify c-Cbl (henceforth referred to as Cbl) as a GM-CSF receptor (GMR) adaptor protein that targets Src for ubiquitin-mediated destruction upon GM-CSF stimulation and show that a loss of negative regulation of Src is pivotal in the hyperactivation of GMR signaling in Cbl-mutated JMML cells. Notably, dasatinib, an U.S. Food and Drug Administration-approved multikinase inhibitor that also targets Src family, dramatically attenuated the spontaneous and GM-CSF-induced hypersensitive growth phenotype of mononuclear cells from peripheral blood and bone marrow collected from JMML patients harboring Cbl or other known JMML-associated mutations. These findings reveal Src kinase as a critical oncogenic driver underlying JMML.

PAR-1 Mediates the Thrombin-induced Mesothelial Cell Overproduction of VEGF and PAI-1

Thrombin mediates an excess in the production of neoangiogenetic (VEGF) and profibrotic (PAI-1) factors in human peritoneal mesothelial cells (HMC). The mechanisms leading to this overproduction have not been elucidated so far; in the context of peritoneal dialysis it can result in impaired peritoneal membrane function. This study was performed to evaluate the presence of the thrombin receptor protease-activated receptor-1 (PAR-1) in HMC and to characterize its function in the thrombin-dependent effects mentioned above. All experiments were performed using cultured primary HMC. Real-Time PCR and Western Blot were used to evaluate PAR-1; ELISA and Real-Time PCR were employed to examine PAR-1 effects on target mediators. We found that cultivated primary HMC show a basal presence of PAR-1. Stimulation with IL-1β induced an increase of the mesothelial PAR-1 expression whereas stimulation with glycosilated human serum albumin or the ligand thrombin itself resulted in decreased PAR-1 expression. Stimulation with the specific PAR-1 ligand TFLLR-NH(2) caused increased VEGF and PAI-1 levels similar to stimulation with thrombin, whereas preincubation with PAR-1 blocking antibodies ATAP2 and WEDE15 attenuated the thrombin-induced overproduction of VEGF and PAI-1. HMC express PAR-1 and the receptor is involved in thrombin effects on these cells. These findings may be a basis for pharmacological prevention of neoangiogenesis and adhesions in the context of peritoneal dialysis and peritonitis.

GDF5 Regulates TGFß-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGFß Peptides

BackgroundTGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß.MethodsWe have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis.ResultsTGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis.ConclusionsTGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.

Critical Role of the Gab2/PI3K/mTOR Pathway in the Pathogenesis of Ptpn11 (Shp2) Mutation-Induced Myeloproliferative Disease.

Abstract 2856▪▪This icon denotes a clinically relevant abstractGermline and somatic mutations (heterozygous) in Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, have been identified in juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disease (MPD), and pediatric acute leukemias. These mutations cause hyperactivation of Shp2 catalytic activity and enhance the binding of mutant Shp2 to signaling partners. Ptpn11 mutations are sufficient to drive the development of JMML-like MPD and acute leukemias in mice, suggesting that they play a causal role in the pathogenesis of hematological malignancies. However, the mechanisms by which Ptpn11 mutations induce these malignancies are not completely understood and the signaling partners that mediate the pathogenic effects of Ptpn11 mutations have not been explored. We previously generated a line of conditional knock-in mice with Ptpn11E76K mutation, the most common and most active Ptpn11 mutation found in JMML and acute leukemias. Induced knock-in of this mutation in hematopoietic cells resulted in MPD with full penetrance as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors (J. Exp. Med., 2011). Recently, we discovered that the interaction between Shp2 E76K and Gab2, a prominent interacting protein of Shp2 and a scaffolding protein important for cytokine-induced PI3K/Akt/mTOR signaling, was greatly enhanced, and that mTOR was highly activated in Ptpn11E76K/+ MPD cells. To address the role of Gab2 and mTOR in the pathogenesis of Ptpn11E76K/+ mutation-induced MPD, Ptpn11E76K/+/Gab2-/- double mutant mice were generated and their phenotypes were compared with those of Ptpn11E76K/+ single mutant mice. MPD phenotypes were markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice. Overproduction of myeloid cells in the bone marrow was alleviated, and splenomegaly was diminished in the double mutants. Myeloid cell infiltration in the liver also decreased. Cytokine (IL-3 and GM-CSF) sensitivity of myeloid progenitors was significantly decreased in Ptpn11E76K/+/Gab2−/− mice as compared to that in Ptpn11E76K/+ mice. Hyperactivation of HSCs and excessive myeloid differentiation caused by Ptpn11E76K mutation were largely corrected by deletion of Gab2. Furthermore, we treated Ptpn11E76K/+ mice with Rapmycin, a specific and potent mTOR inhibitor, which substantially diminished MPD phenotypes. Collectively, this study reveals the essential role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic effects of Ptpn11E76K/+ mutation and suggests that Gab2 and mTOR are potential therapeutic targets for the treatment of Ptpn11-associated hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

A Functional Thrombopoietin Receptor Is Required for Full Expression of Phenotype in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera

Abstract 427Polycythemia vera (PV) is a clonal stem cell disorder in which there is overproduction of red cells, white cells and platelets, extramedullary hematopoiesis and transformation to myelofibrosis. Enhanced hematopoiesis in PV is due to constitutive activation of JAK2, the cognate tyrosine kinase of type I cytokine receptors such as the erythropoietin and thrombopoietin receptors. Hematopoietic stem cells express the thrombopoietin receptor (Mpl) but paradoxically mice lacking the MPL gene exhibit only thrombocytopenia while maintaining the capacity to not only substantially elevate their platelet and leukocyte counts during marrow reproductive stress but also the number of multipotent and committed hematopoietic progenitor cells as well. Equally paradoxical, Mpl expression is impaired in PV, but how impaired expression of a master regulator of stem cell function could be associated with a global increase in hematopoiesis has been a conundrum. Recently, data were presented (Blood 119: 4625, 2012) suggesting that Mpl down regulation in PV was due to constitutive JAK2 activation, which abrogated the antiproliferative effects of the high plasma thrombopoietin (TPO) levels in PV, thus sustaining the PV phenotype. We have used a JAK2 V617F transgenic mouse model (V617Ftg) (Blood 111:5109, 2009) to examine the role of Mpl in the pathogenesis of the JAK2 V617F-positive PV phenotype and have observed that deletion of the MPL gene ameliorated the PV phenotype in this model, reducing the hemoglobin level, leukocyte and platelet counts and splenomegaly and reversing marrow fibrosis, while the presence of a single MPL gene was sufficient to restore the PV phenotype. However, total deletion of the MPL gene does not perfectly model human PV where Mpl is present but quantitatively reduced. Therefore, we examined the effect of manipulating Mpl behavior in vivo by modifying Mpl signaling while maintaining Mpl expression, first, by eliminating TPO production, and second, by expressing a functionally impaired Mpl receptor, and as a control, specifically impairing megakaryocyte differentiation alone. To this end, we crossbred the V617Ftg PV mouse with a TPO gene knockout mouse (TPO ko), with a mouse homozygous for the MPL mutation, C40R (C40R hom), which in the homozygous state causes thrombocytopenia, and finally, with an MKL1 knockout mouse (MKL1ko) that is thrombocytopenic due to impaired megakaryocyte maturation. Crossbreeding produced the three expected genotypes (JAK2 V617F/TPO ko, JAK2 V617F/C40R hom and JAK2 V617F/MKL1ko) without unexplained mortality or impoverished growth. We compared the blood counts, spleen weights and bone marrow and spleen histology of these three genotypes with each other and with JAK2 V617F/TPO heterozygous (het) mice over a 33 week period. The natural history of the V617Ftg PV mouse is one of initial erythrocytosis, thrombocytosis, leukocytosis, splenomegaly, and expansion of short term marrow repopulating cells (ST-HSC), common myeloid progenitor cells (CMP) and marrow and spleen committed hematopoietic progenitor cells (HPC)(CFU-GEMM, CFU-GM, BFU-E, CFU-E and CFU-MK), and myelofibrosis with a subsequent decline in erythrocytosis. Abrogating TPO production in the V617Ftg PV mouse normalized the platelet and leukocyte counts, delayed the development of erythrocytosis, reduced spleen size and ST-HSC, CMP, and all marrow and spleen committed HPC compartments and reversed marrow fibrosis. The V617Ftg PV - C40R MPL cross prevented thrombocytosis, erythrocytosis, leukocytosis and splenomegaly, and reduced all the marrow and spleen committed HPC compartments. By contrast, the V617Ftg PV-MKL1 ko cross slowed the rate of rise of the platelet count and hemoglobin level but had no effect on splenomegaly or marrow fibrosis, nor was there a reduction in any of the marrow or spleen committed HPC. Expression of one TPO gene by crossing the V617Ftg PV mouse with mice heterozygous for the TPO gene (JAK2 V617F/TPO het) restored the PV phenotype. In conclusion, while it is unquestioned that JAK2 V617F is necessary for expression of the PV phenotype, our data indicate that it alone is insufficient and requires not only the expression of a functional TPO receptor but also receptor activation by its cognate ligand TPO. This latter observation suggests that interfering with the TPO-Mpl interaction could be of therapeutic value in the treatment of human PV. Disclosures:No relevant conflicts of interest to declare.

Endoplasmic reticulum stress associated retinal photoreceptor cell death in the transgenic mutant rhodopsin S334ter-3 rats

Retinitis pigmentosa (RP) is associated with the expression of a mutant photoreceptor-specific protein. We hypothesized that missorted mutant rhodopsin induces an evolutionarily conserved compensatory mechanism known as the unfolded protein response (UPR). Although, the endoplasmic reticulum (ER) stress is a protective cellular response against stresses, chronic ER stress activates a UPR-dependent death pathway that results in retinal degeneration. The purpose of this study was to determine whether ER stress is one of the causes for photoreceptor cell death. Here, we used retinal 661W cells and transgenic mutant rhodopsin S334ter-3 and S334ter-4 rats. The ER stressors such as homocysteine and calcimycin activated the UPR-specific proteins, ATF4, Bip, CHOP, and procaspase-12; caused overproduction of ROS; and induced cell death in cultured retinal 661W cells. Similarly, retinas of transgenic S334ter-3 but not S334ter-4 rats revealed the activation of UPR-specific proteins Bip, ATF4, CHOP, calnexin, and procaspase-12. Retinal slices of S334ter-3 rats at postnatal day 5 exhibited overproduction of ROS and dead cells in the photoreceptor cell layer. Thus, the activated apoptotic UPR in retinal photoreceptor cells of transgenic mutant rhodopsin S334ter-3 rats suggested that UPR might play a role in retinal photoreceptor cell death in S334ter-3 rats.

Scutellaria baicalensis Alleviates Cantharidin-Induced Rat Hemorrhagic Cystitis through Inhibition of Cyclooxygenase-2 Overexpression

Cantharidin, an active component in mylabris, is used in traditional Chinese medicine (TCM) to treat scabies and hepatoma, but accompanied by hemorrhagic cystitis. Evidence shows that cantharidin induces human bladder carcinoma cell death through COX-2 overexpression in vitro. In TCM, Scutellaria baicalensis is usually used to cure mylabris-induced hematuria. This work was undertaken to determine the mechanisms of cantharidin-induced rat hemorrhagic cystitis and explore the uroprotective effect of S. baicalensis. In vitro results showed cantharidin could induce cytotoxicity through prostaglandin (PG)E2 overproduction of T24 cells. Boiling-water extract of S. baicalensis (SB-WE) could significantly inhibit PGE2 production and COX-2 expression in lipo-polysaccharide-induced RAW 264.7 cells, indicating obvious anti-inflammatory abilities. In vivo results indicated that cantharidin caused rat hemorrhagic cystitis with hematuria via c-Fos and COX-2 overexpression. SB-WE was given orally to cantharidin-treated rats, whereby hematuria level, elevated PGE2 and COX-2 protein overexpression were significantly and dose-dependently inhibited by SB-WE. The anti-inflammatory components of SB-WE are baicalin and wogonin, whose contents were 200.95 ± 2.00 and 31.93 ± 0.26 μg/mg, respectively. In conclusion, cantharidin induces rat cystitis through c-Fos and COX-2 over-expression and S. baicalensis can prevent the resulting hematuria because of its anti-inflammatory effects.

Squamous Cell Carcinoma Accompanying Chronic Myeloid Leukemia: Case Report and Review of the Literature

DOI: 10.7247/jiumf.2012.19.1.40_45 Background: Chronic Myelogenous Leukemia (CML) is a myeloproliferative disorder characterized by, overproduction of myeloid cells. Hydroxyurea is an antimetabolite which is commonly used in myeloproliferative syndromes. Reported skin changes related to hydroxyurea therapy may range form xerosis to mucocutaneous cancers. Aim: In this study, our aim was to understand and discuss the underlying reason for the development of squamous cell carcinoma (SCC) in patients recieving hydroxyurea and present a case, in whomwe operated to excise the tumoral masses and reconstruct them. Materials and Methods: After recieving hydroxyuea therapy for four years, the patient was diagnosed as squamous cell carcinoma on the upper lip and, on the dorsum of the left hand. She was operated and the tumoral masses were excised. The lesion on the upper lip was reconstructed with nasolabial flap, inner and inferior sides of the flap was skin grafted. The defect on the dorsum of the hand was skin grafted. Results: No complication related to operation occured. Throughout postoperative eight months of follow-up period, the patient had no complaint regarding the operation. Conclusion: Skin lesions of patients recieving hydroxyurea therapy must be carefully examined and monitored regularly, since these lesions may proceed to skin cancers.