Caracterização de uma nova proteina com repetições de anquirina, ANKHD1, na hematopoese normal e leucemica

Orientador: Sara Teresinha Olalla SaadTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: Importante passo para a compreensão dos processos fisiopatológicos das neoplasias é a identificação de genes ativamente expressos e das funções biológicas de cada proteína codificada por estes genes. Ankyrin Repeat Single KH Domain containing 1 (ANKHD1) foi inicialmente identificada em células de adenocarcinoma de próstata humano (LNCaP), no ano de 2003. Entretanto, seu padrão de expressão e sua função ainda não haviam sido caracterizados. A ANKHD1 é uma proteína ortóloga à Multiple Ankyrin repeat and single KH domain (Mask) da Drosophila melanogaster. Mask foi identificada através de um rastreamento genético utilizado para detectar novas proteínas associadas à proteína tirosina fosfatase Corkscrew (CSW), homóloga à Src Homology-2 domain-containing protein tyrosine Phosphatase-2 (SHP2) humana. SHP2 é uma fosfatase de tirosina citoplasmática codificada pelo gene PTPN11 e exerce papel fundamental no desenvolvimento da hematopoese normal e leucêmica. Os objetivos gerais do presente estudo foram caracterizar o padrão de expressão gênica e protéica de ANKHD1 em células hematopoéticas normais e leucêmicas e sua participação nas vias de sinalização celular. Neste estudo, foi demonstrada a elevada expressão gênica e protéica de ANKHD1 em linhagens de leucemias agudas humanas (KG-1, HEL, K562, NB4, HL-60, Jurkat, MOLT4, Raji, Daudi e Namalwa) e em amostras de 38 pacientes com diagnóstico de leucemia aguda, quando comparadas às células hematopoéticas normais. A expressão protéica de ANKHD1 em diferentes tecidos humanos normais (rim, baço, estômago, intestino delgado, músculo esquelético, fígado, pulmão e linfonodo) foi detectada em intensidades variáveis. A associação da ANKHD1 com SHP2 foi identificada, através de Western Blotting, em células de leucemia mielóide crônica em fase blástica (K562) e células LNCaP. Entretanto, esta associação não foi detectada nas linhagens leucêmicas KG1, HL60, Daudi e Jurkat. A ANKHD1 foi localizada no citoplasma de células hematopoéticas normais e leucêmicas. Detectou-se a fosforilação de ANKHD1 em serina em células leucêmicas, mas não em células hematopoéticas normais. Através de ensaio de duplo híbrido em levedura, utilizando-se uma biblioteca de cDNA de medula óssea humana normal, detectou-se a interação de ANKHD1 com as proteínas SIVA1 e SIVA2, proteínas pró-apoptóticas altamente expressas em linhagens de leucemia linfóide aguda. Análises preliminares indicaram que a inibição da expressão protéica de ANKHD1, através de RNAi, induziu à apoptose celular em células leucêmicas, sugerindo uma função anti-apoptótica à ANKHD1. Em conclusão, o presente estudo identificou ANKHD1 como uma nova proteína altamente expressa em leucemias agudas, associada à SHP2 e SIVA em diferentes células, e, possivelmente, envolvida com o fenótipo anormal da célula leucêmica através de uma função anti-apoptótica. Os achados aqui descritos sugerem que ANKHD1 pode ser uma molécula alvo para a terapia da leucemia no futuro, e permitirão direcionar novos estudos com o objetivo de melhor elucidar as funções específicas de ANKHD1 em diferentes células hematopoéticas normais e leucêmicasAbstract: One step in the path towards building a comprehensive molecular portrait of human cancer is the definition of actively expressed genes and the function of their coding proteins. The Ankyrin Repeat Single KH Domain containing 1 protein (ANKHD1) was first described in humans in a prostate carcinoma cell line LNCaP, in 2003; however, its expression pattern and its function have not yet been described. ANKHD1 is an orthologous protein of the Drosophila melanogaster, MASK (Multiple Ankyrin repeat and single KH domain), where it was first identified using a genetic screen designed to discover proteins that interact with the protein tyrosine phosphatase Corkscrew (CSW), which is a homolog to the SH2-containing protein tyrosine phosphatase (SHP2) in humans. SHP2 is a cytoplasmic protein-tyrosine phosphatase, coded by the PTPN11 gene and plays an important role in the development of normal hematopoiese and leukemogenesis. The aim of the present study was to characterize the gene and protein expression pattern of ANKHD1 in normal hematopoietic cells and in leukemia cells, and its role in signaling pathways. In the present study, the overexpression of ANKHD1 mRNA and its protein was demonstrated in human leukemia cell lines (KG-1, HEL, K562, NB4, HL-60, Jurkat, MOLT4, Raji, Daudi and Namalwa) and in 38 patients with a diagnosis of acute leukemia, compared to normal hematopoietic cells. The ANKHD1 protein was found to have a variable expression in different normal tissues (kidney, spleen, stomach, small intestine, skeletal muscle, liver, lung and lymph node). An association of ANKHD1 and SHP2 was found, through imunopreciptation and Western Blotting, in the chronic myeloid leukemia blastic phase cell line (K562) and in LNCaP cells. However, this association was not detected in the leukemia cell lines, KG1, HL60, Daudi and Jurkat. ANKHD1 was found located in the cytoplasm of normal hematopoietic cells and leukemia cells. ANKHD1 was found to be phosphorylated at serine in leukemic cells, but not in normal hematopoieitic cells. Through the yeast two-hybrid system, using a cDNA library from normal human bone marrow, the interaction of ANKHD1 with SIVA1 and SIVA2 was detected. SIVA isoforms are pro-apoptotic proteins, overexpressed in acute lymphoblastic leukemia cell lines. Preliminary studies showed that the inbition of ANKHD1 expression, through RNAi, resulted in increased apoptosis in leukemic cells, which suggests an anti-apoptotic function for ANKHD1. In conclusion, the present study identified ANKHD1 as a new protein that is overexpressed in leukemic cells. The protein interacts with SHP2 and SIVA in different cells and is probably associated with the abnormal phenotype of the leukemia cell through its anti-apoptotic function. These findings suggest that ANKHD1 may be a molecular target for a rational therapy for leukemia in the near future, and open a new field of investigation to better define the specific roles of ANKHD1 in different normal and leukemic hematopoietic cellsDoutoradoBiologia Estrutural, Celular, Molecular e do DesenvolvimentoDoutor em Fisiopatologia Medic

ANKHD1 (ankyrin repeat and KH domain containing 1)

ANKHD1 is differentially expressed in human cancers, and potentially regulates multiple cellular functions and participates as a scaffold for protein-protein interactions through its ankyrin-repeat domains. Recently, studies have indicated that ANKHD1 is involved in the regulation of important biological process that participate in the malignant phenotype, including cell cycle progression, proliferation, clonogenicity and migration. The present review on ANKHD1 contains data on DNA/RNA, protein encoded and where the gene is implicated

IRS2 (insulin receptor substrate 2)

Insulin receptor substrate 2 (IRS2) belongs to the insulin receptor substrate protein family and was initially discovered as an alternative route for signaling mediated by the insulin receptor. Currently, IRS2 has been well-established to mediate mitogenic and antiapoptotic signaling from several important cellular receptors. In the last years, many studies have indicated that IRS2 participates in the regulation of important biological processes involved in cancer phenotype, including cell proliferation, clonogenicity, metabolism and survival. The present review contains data on IRS2 DNA/RNA, protein encoded and function

Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. in this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL in conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells. (C) 2011 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Estadual Campinas, Hematol & Hemotherapy Ctr, UNICAMP, Hemoctr,Inst Nacl Ciencia & Tecnol Sangue, BR-13083878 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

Low Bone Mass Density Is Associated With Hemolysis In Brazilian Patients With Sickle Cell Disease.

To determine whether kidney disease and hemolysis are associated with bone mass density in a population of adult Brazilian patients with sickle cell disease. Bone involvement is a frequent clinical manifestation of sickle cell disease, and it has multiple causes; however, there are few consistent clinical associations between bone involvement and sickle cell disease. Patients over 20 years of age with sickle cell disease who were regularly followed at the Hematology and Hemotherapy Center of Campinas, Brazil, were sorted into three groups, including those with normal bone mass density, those with osteopenia, and those with osteoporosis, according to the World Health Organization criteria. The clinical data of the patients were compared using statistical analyses. In total, 65 patients were included in this study: 12 (18.5%) with normal bone mass density, 37 (57%) with osteopenia and 16 (24.5%) with osteoporosis. Overall, 53 patients (81.5%) had bone mass densities below normal standards. Osteopenia and osteoporosis patients had increased lactate dehydrogenase levels and reticulocyte counts compared to patients with normal bone mass density (p<0.05). Osteoporosis patients also had decreased hemoglobin levels (p<0.05). Hemolysis was significantly increased in patients with osteoporosis compared with patients with osteopenia, as indicated by increased lactate dehydrogenase levels and reticulocyte counts as well as decreased hemoglobin levels. Osteoporosis patients were older, with lower glomerular filtration rates than patients with osteopenia. There was no significant difference between the groups with regard to gender, body mass index, serum creatinine levels, estimated creatinine clearance, or microalbuminuria. A high prevalence of reduced bone mass density that was associated with hemolysis was found in this population, as indicated by the high lactate dehydrogenase levels, increased reticulocyte counts and low hemoglobin levels.66801-

Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

<p>Abstract</p> <p>Background</p> <p>Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease.</p> <p>Methods</p> <p>In this study, gene expression profiles of CD34⁺cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts.</p> <p>Results</p> <p>In CD34⁺cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts.</p> <p>Conclusions</p> <p>These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34⁺cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.</p

PTK2 and PTPN11 expression in myelodysplastic syndromes

OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors

De novo AML exhibits greater microenvironment dysregulation compared to AML with myelodysplasia-related changes

The interaction between the bone marrow microenvironment and malignant hematopoietic cells can result in the protection of leukemia cells from chemotherapy in both myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We, herein, characterized the changes in cytokine expression and the function of mesenchymal stromal cells (MSC) in patients with MDS, AML with myelodysplasia-related changes (MRC), a well-recognized clinical subtype of secondary AML, and de novo AML. We observed a significant inhibitory effect of MDS-MSC on T-lymphocyte proliferation and no significant differences in any of the cytokines tested. AML-MSC inhibited T-cell proliferation only at a very low MSC/T cell ratio. When compared to the control, AML-MRC-derived MSC presented a significant increase in IL6 expression, whereas de novo AML MSC presented a significant increase in the expression levels of VEGFA, CXCL12, RPGE2, IDO, IL1 beta, IL6 and IL32, followed by a decrease in IL10 expression. Furthermore, data indicate that IL-32 regulates stromal cell proliferation, has a chemotactic potential and participates in stromal cell crosstalk with leukemia cells, which could result in chemoresistance. Our results suggest that the differences between AML-MRC and de novo AML also extend into the leukemic stem cell niche and that IL-32 can participate in the regulation of the bone marrow cytokine milieu.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Univ Estadual Campinas, Hematol & Transfus Med Ctr, Hemoctr Unicamp, Inst Nacl Ciencia & Tecnol Sangue, Sao Paulo, BrazilUniv Sao Paulo, Ribeirao Preto Med Sch, Dept Internal Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilFed Univ Vale do Sao Francisco, Paulo Afonso, BA, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilWeb of Scienc

Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic proteins that integrate and coordinate the transmission of signals from the extracellular to the intracellular environment via transmembrane receptors, thus regulating cell growth, metabolism, survival and proliferation. The PI3K/AKT/mTOR and MAPK signaling pathways are the best-characterized downstream signaling pathways activated by IRS signaling (canonical pathways). However, novel signaling axes involving IRS proteins (noncanonical pathways) have recently been identified in solid tumor and hematologic neoplasm models. Insulin receptor substrate-1 (IRS1) and insulin receptor substrate-2 (IRS2) are the best-characterized IRS proteins in hematologic-related processes. IRS2 binds to important cellular receptors involved in normal hematopoiesis (EPOR, MPL and IGF1R). Moreover, the identification of IRS1/ABL1 and IRS2/JAK2V617F interactions and their functional consequences has opened a new frontier for investigating the roles of the IRS protein family in malignant hematopoiesis. Insulin receptor substrate-4 (IRS4) is absent in normal hematopoietic tissues but may be expressed under abnormal conditions. Moreover, insulin receptor substrate-5 (DOK4) and insulin receptor substrate-6 (DOK5) are linked to lymphocyte regulation. An improved understanding of the signaling pathways mediated by IRS proteins in hematopoiesis-related processes, along with the increased development of agonists and antagonists of these signaling axes, may generate new therapeutic approaches for hematological diseases. The scope of this review is to recapitulate and review the evidence for the functions of IRS proteins in normal and malignant hematopoiesis