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Introduction: Satisfactory response is present for the majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP) treated with tyrosine kinase inhibitors (ITK) . However, some pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The oxidative stress modulation is tightly related with the physiopathology of various hematologic diseases and can cause cell death, apoptosis and necrosis. Peroxiredoxins (Prdx) are a family of multifunctional antioxidant thioredoxin-dependent peroxidases that protect cells against oxidative stress and modulate signaling cell proliferation pathways and may influence the metabolism of ITKs.The aim of this study was to analyze PRDX1, PRDX2 and PRDX6 levels of CML pts and correlate with cytogenetics and molecular responses. Methods: PRDX1, PRDX2 and PRDX6 expression was evaluated in 20 blood donors, 18 newly diagnosed CML pts and 22 previously treated pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis or during treatment and RNA samples were submitted to the synthesis of complementary DNA (cDNA) using the kit RevertAid™ HMinus First Strand cDNA Synthesis Kit (Fermentas, Life Sciences). For cDNA synthesis, 3 ug of RNA was used and peroxiredoxins expression was evaluated by real-time PCR with Syber Green (Applied Biosystems) and endogenous (β-Actina and GAPDH) controls. The results were analyzed using 2-ΔΔCT. Statistical analysis were made by using Mann Withney’s T test. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RQ-PCR. Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS). Results: 40 CML pts, 55% male, median age of 53 years (23-84) were evaluated, 60% in chronic phase (CP), 30% in accelerated phase (AP) and 10% in blast crisis (BC). The mean of PRDX transcript levels in the total group was (PRDX1: 0.006 and 10.10 / PRDX2: 0.002 and 16.26 / PRDX6: 0.003 and 49.97) respectively (PRDX1: 1.2 / PRDX2: 0.9 / PRDX6: 15.36). The results showed that there are a significantly difference (p Conclusion:Is known that that the increase of ROS in CML leads to an increase of DNA damage, triggering genomic instability and resulting in accumulation of mutations and chromosomal aberrations, and contribute to the mechanism of acquisition of resistance to TKI inhibitors. The decrease of Peroxiredoxins expression observed in the responsive group, could contribute to this process, since the detoxification of these species are compromising and the effects caused by oxidative stress are even more drastic, leading to mutations that could be followed by TKi resistance. The relation between Prdx and CML not yet been elucidated. Disclosures No relevant conflicts of interest to declare.

Abstract 5125 The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 4415 The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p Disclosures: No relevant conflicts of interest to declare.

Abstract 4797 Background: Extensive studies have led to a considerable understanding of the cellular and molecular control of haemoglobin production during red blood cell differentiation, however, identification of the genes expressed as part of the erythroid differentiation programme remains an important goal because of the insights that these data will bring to erythrocyte biology and disease. Previous results using SAGE identified 93 differentially-expressed genes during erythroid development. One of these genes, EYA3, a homologue gene of Eyes Absent 3 in Drosophila, is a transcription cofactor with intrinsic phosphatase activity and its expression was observed to be high at the end of CD34+ cell differentiation and in human bone marrow. Aim: To evaluate globin gene, fetal hemoglobin (HbF) expressions and apoptosis levels in the erythroleukemic K562 cell line after EYA3 gene silencing and induction with hemin. Methods: Four different cultures from human K562 cells (1×105cells/mL in DMEM, 10% FBS, penicillin/streptomycin, 5% CO2, 37°C) were transfected with control or EYA3 knockdown lentiviruses (MOI=2.5). After proliferation and selection of successfully transfected cells with puromicin (2.0 ug/mL), cells were treated with 30μM hemin and collected after 0, 24, 48, 72 and 96h for gene expression and flow cytometry analyses. EYA3, LXN, α, and g-gene expression was measured by qRT-PCR and normalized using the Genorm program. HbF expression and apoptosis were evaluated by flow cytometry. Results: Analysis of globin gene expression showed that α-globin gene was downregulated in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (1.791±0.1735; 0.7404±0.1709, respectively, **P Conclusions: Our results show that silencing EYA causes modifications in the expression pattern of α- and g-globin gene expression as well as in HbF expression pattern and apoptosis levels in a model of erythroid differentiation. Further studies should be performed in primary erythroid cell cultures using siRNA-based gene silencing and overexpression of these genes to determine how these genes are involved in the mechanisms of globin gene regulation. Support by FAPESP, CNPq and INCTS Disclosures: No relevant conflicts of interest to declare.

Abstract 3195 The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies. Support by FAPESP, CNPq.and INCTS Disclosures: No relevant conflicts of interest to declare.

Abstract 2066 Hereditary persistence of fetal hemoglobin (HPFH) is a condition that prevents hemoglobin switching and the consequent silencing of the gamma globin genes, resulting in continued hemoglobin (Hb) F synthesis in adults. Two types of HPFH are responsible for this phenotype: deletional HPFH – deletions in the end of the beta globin locus – and non-deletional HPFH (ndHPFH) – single point mutations in the proximal promoter of both gamma globin genes. Sickle cell anemia patients or beta-thalassemia patients that present HPFH show high levels of HbF that are associated with less severe clinical course in these diseases. The development of new therapies based on the reactivation of gamma globin expression may be important for the treatment of these patients. The Brazilian ndHPFH type is characterized as a C→G substitution in the A gamma globin promoter at position –195 and the molecular mechanism responsible for the reactivation of this gene in the Brazilian ndHPFH type remains unclear. In contrast to the British ndHPFH type (-198), where the mechanism responsible for the increase of HbF levels is mediated by the raising in the affinity for the Sp1 transcription factor (TF), the Brazilian ndHPFH mutation does not affect Sp1 binding. Thus, other TF may be involved in the reactivation of the A gamma globin gene in the Brazilian ndHPFH type. The aim of this study was to investigate the mechanism involved in the reactivation or repression of the A gamma globin gene in the Brazilian ndHPFH type and identify possible TF responsible for this phenotype. In vitro primary human erythroblast cultures, derived from human CD34+ hematopoietic cells from 4 Brazilian ndHPFH type subjects and 4 control subjects, were proliferated and differentiated into late stage erythroblasts. The nuclear extracts from predominantly basophilic and polychromatic erythroblasts were used to profile TF activity using Protein-DNA Array method. The analysis of the array densitometry identified a number of TF whose DNA binding activities were either enhanced or repressed in the Brazilian ndHPFH cultures. Among the TF analyzed, the NF-E1/YY1 and the PAX-1 were selected for this study. Since this assay requires a secondary method to confirm these results, nuclear extracts were used to conduct chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). ChIP was carried out using antibodies against NF-E1/YY1 and PAX-1 to quantify the binding to these TF to the –195 A gamma globin promoter region. EMSA was performed using probes with the same sequence spotted on the array membrane to analyze the activity of NF-E1/YY1 and PAX-1. Both methods confirmed and validated the previous array results. NF-E1/YY1 is a transcription factor that represses embryonic (epsilon) and fetal (gamma) globin genes. Protein-DNA array and EMSA showed a decreased binding of NF-E1/YY1 in Brazilian ndHPFH nuclear extracts and ChIP analysis revealed diminished NF-E1/YY1 occupancy at the –195 A gamma globin promoter region of Brazilian ndHPFH. The consensus binding site for NF-E1/YY1 is a CCAN motif that is observed between the –195 and –192 position in the A gamma globin promoter region. The C→G substitution at –195 position may disrupt this DNA binding site, cause decreased NF-E1/YY1 interaction and probably allows the binding of PAX-1, a transcriptional activator with a paired box DNA-binding domain that has as a DNA binding core motif, the sequence TTCCGC. This sequence, located between the –199 and –194 position in the A gamma globin promoter, is only presente in the Brazilian type of ndHPFH. Our protein-DNA array and EMSA results showed an increased binding of PAX-1 in the Brazilian ndHPFH nuclear extracts and quantitative ChIP analysis with anti-PAX-1 antibody showed that PAX-1 binds to the –195 A gamma globin promoter region only in the presence of this C→G substitution. These results suggest that the –195 site (C→G) in the A gamma globin promoter region may decrease NF-E1/YY1 binding and increase PAX-1 binding in this DNA region, probably resulting in the reactivation of the A gamma globin gene. The increase in the HbF levels in the Brazilian ndHPFH occurs differently from the British ndHPFH type and represents a novel mechanism of A gamma globin reactivation. Such findings may lead to the development of future therapeutic strategies for HbF induction in the treatment of other hemoglobinopathies. Support by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

Abstract 2215 Poster Board II-192 The exact action mechanism of the protein BCR-ABL is unknown, studies in vitro and in animal models showed that the activity of protein tyrosine kinase (TK), by itself, is sufficient to cause the development of CML. Thus, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. The use of methods to detect the difference in gene expression between CML and normal granulocytes could bring new insights in the understanding of these complex mechanisms, highlighting new therapeutic targets for the CML treatment. Evaluation of the differential gene expression between CML and normal granulocytes using the Subtractive Suppression Hybridization (SSH) and investigation of the involvement of a set of genes in modulating the development of CML. The SSH libraries were constructed using a pool of granulocytes RNA's extracted from CML patients and from healthy blood donors. Real-Time (RT-PCR) was used to validate the results and evaluation of gene expression in granulocytes and in mononuclear cells in the pool of patients and controls used for the construction of libraries. The expression of the RUNX1 gene, whose expression was the most differential between the libraries, was evaluated in mononuclear cells of patients with CML using RT-PCR. The comparison between granulocytes of CML patients and control granulocytes showed 39 genes exclusively expressed in CML and 169 genes in controls. For validation, we evaluated the expression of genes RUNX1, KPNA6, TOB1, SEPT5, CDC42SE, ITCH, MIER and GP1BA by RT-PCR in the pool of patients and controls used for the construction of libraries. Among these genes, the RUNX1 gene showed substantial difference, and was also studied in mononuclear cells from 20 patients with CML in different stages of the disease and using different types of treatment with TK inhibitors. The expression of this gene was highly altered in patients in advanced phase of disease when compared with patients who are responding to treatment. The genes identified in this study may be related to the development and progression of CML. Among them, the RUNX1 gene was shown to be one of the most promising, since the occurrence of chromosomal translocations in this gene has been associated with different types of acute leukemia – however its association with CML is not yet clear. The results showed a relationship between the expression of this gene and progression of the disease, beyond the identification of several genes that may lead to a better understanding of CML and help in identifying new therapeutic targets. This work was supported by FAPESP. Disclosures: Off Label Use: Development of TKI in the treatment of CML.

Erythroid differentiation is a dynamic and complex process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. Extensive studies have led to a considerable understanding of the cellular and molecular control of hemoglobin production during red blood cell differentiation, however, a complete understanding of human erythropoiesis will require a robust description of the entire transcriptome of these cells during differentiation. From a global point of view of cell metabolic regulation, where genomic information could be complemented with gene expression, the use of methods that enable quantification of the entire transcriptome of the red blood cell during differentiation is of great importance. In this study, the gene expression profiles during differentiation of Human erythroid cells of a normal blood donor in a two-phase liquid culture (Fibach & Rachmilewitz, 1993) were evaluated using Serial analysis of gene expression (SAGE). Global gene expression was evaluated in cells collected immediately before the addition of erythropoeitin (0 hour) and 192 and 336 hours after the addition of this hormone. We generated a total of 30512 tags at 0h, 30117 tags at 192h and 30189 tags at 336h, representing 12026, 11709 and 11337 unique tags, respectively. In the 0h library, a high expression of ferritin genes and CD74 antigen gene was observed. As expected, the expression of globin genes started during intermediate stages of differentiation (predominantly basophilic erythroblasts) and were the most expressed genes at the end of the culture (predominantly orthocromatic erythroblasts). Ribosomal genes were the most expressed genes at 192 hours, indicating an increase in protein synthesis. To identify the genes that were differentially expressed between the libraries, a P value < 0.01 and fold ≥ 5 were considered as statistically significant. In the comparison of the 0h and 192h libraries, 179 differentially expressed transcripts were identified. From these genes, in addition to the globin genes, we found an up-regulation of several genes related to protein binding (LXN, GSTM3, and TRIP6), transcription factor (GATA-1), hydrolase activity (TPSAB1), ion transport (SLC12A9) and regulation of apoptosis (PRDX2). Comparing the 192h and 336h libraries, 103 differentially expressed transcripts were identified. The up-regulated genes were generally related to hemoglobin synthesis, such as ALAS2, involved in the biosynthesis of the heme group or related to intracellular transport such as MSCP and NUDT4 and cell differentiation such as GDF15. The transcription of some of these genes, such as SLC12A9, TRB3, EYA3 and TWIST2 are described for the first time during erythroid differentiation. The results indicated that the global aspects of the transcriptome were similar during differentiation for the majority of the genes and that probably a relative small set of genes is involved in the modification of erythroid cells during differentiation. The results of this study amplify the previous published data (Komor et al, 2005) and may contribute to the comprehension of erythroid differentiation and identification of new target genes involved in some erythroid diseases.

Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

The term, hereditary persistence of fetal hemoglobin (HPFH), describes a hereditary benign disease, characterized by an increase in fetal hemoglobin (HbF) during adult life. Non-deletional forms of HPFH are characterized by single-base mutations in the promoter region (most of them between −114 and −202 from the cap site) of either the Gγor Aγ-globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ-195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B). A previous study showed that the -195 mutations alone was not able to increase gene expression in vitro and a modest increase was observed when a LCR element fragment (HS2) was introduced in the construction. This study showed too, that the the mechanism of HbF elevation by the -195 mutation was neither mediated by the Sp-1 transcription factor nor by the creation of a CACCC box, as described for the -198 mutation. Thus, other proteins may be involved in the over-expression of the γ-globin chain and/or may depend on DNA structure and these mechanisms remain to be clarified. To better understand this mechanism we have developed HPFH-B transgenic mice. The promoter with mutation was amplified using the genomic DNA of a HPFH-B patient and cloned in a μLCRAγψβδβ cosmid. This construct, containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into single cell mouse embryos. To detect the differences in developmental regulation of the human gamma-globin gene expression in the transgenic mice, we analyzed the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5 of both mutated and non-mutated transgenic mice by RNAse Protection Assay (RPA) and Real time PCR (RT-PCR). Levels of expression of murine alpha-globin mRNA were used as internal controls in the RPA experiment and levels of murine-GAPDH were used in RT-PCR. mRNA levels of human gamma-globin in fetal liver of transgenic mice containing mutation were clearly higher, as compared with control transgenic mice bearing cosmid construct with wild a type sequence gamma promoter. Additionally a nearly 10-fold increase in human gamma-globin expression was detected in fetal livers of HPFH transgenic mice at E13.5 compared to the yolk sac. Thus, our data indicate that the - 195 mutation is the unique cause of elevation of HbF in Brazilian HPFH, but the exact mechanism needs to be clarified.

Hereditary Persistence of Fetal Hemoglobin is a rare, heterogeneous and benign group of hereditary disorders with an abnormal switch from fetal to adult hemoglobin, resulting in high levels of Hb F in the adult stage. A total of six deletions related to HPFH have been described, associated with increased levels of both gamma chains. Three main hypotheses have been proposed to explain the relationship between these deletions and the non-suppression of gamma genes: the removal of competitive regions that interact with the LCR; the juxtaposition of enhancer elements; and the removal of silencers. Despite evidence to support these hypotheses, however, they are not conclusive. Recently, Xiang and cols (Abstract #1215, 2004 ASH Meeting; Blood, Volume 104, issue 11, November 16, 2004) developed a YAC construct with the whole beta-globin locus containing a deletion of approximately 83.5 Kb responsible for the HPFH-2. Unexpectedly, the gamma gene was completely silenced in the adult transgenic mice. These data suggest that other mechanisms could be involved in the increased levels of HbF in these conditions. The authors speculate that other regions upstream from the cluster may harbor this activity. We, herein, investigate the possible involvement of transcription factors, using the subtractive hybridization method to identify differentially expressed transcripts in reticulocytes from a normal subject and a HPFH-2 subject. We have identified 56 and 106 unique genes in the normal and HPFH-2 cDNA libraries, respectively. Some of these are transcription (zinc fingers and homeobox proteins) and chromatin remodeling (NAP and SWI like proteins) factors that could participate in globin gene regulation. These genes are located in cis or in trans to the deletion and their altered gene expression has been confirmed by Quantitative Real-time PCR in other two HPFH subjects. The data may present new clues about globin gene regulation, the increased expression of gamma gene in deletional HPFH and the dynamic organization of genes and chromosomes in cells.

Hematopoietic stem/progenitor cells with their dual ability for self-renewal, expansion and multilineage differentiation constitute an essential component of hematopoietic transplantation. These qualities, together with their transfection capacity make them useful for gene therapy also. Umbilical cord blood and bone marrow represent efficient sources of these cells. Cord blood stem cells are more primitive than bone marrow cells. They have a greater capacity to self-renewal, proliferation, expansion, multilineage differentiation and are better transfected. They can also be transplanted without complete HLA compatibility. Some recent studies are demonstrating that they could have a better trafficking and homing than the others, which could promote an efficient development and proliferation in the marrow microenvironment and better recovering when transplanted. However, these mechanisms of migration/cell adhesion and cell cycle are not fully understood. The aim of this study is to compare cord blood mononuclear cells with the existing bone marrow mononuclear cells library (esage database) by means of serial analysis of gene expression in order to investigate if there are any differences that could explain those findings. We obtained, by sequencing the SAGE library of cord blood cells, a total of 44.924 tags representing 15.519 unique tags. These unique tags were distribute as: known genes= 7772; sequence predicted or annotated=3935; no matches= 3812. The bone marrow library showed total tags=36577 being unique tags=13075: known genes= 6711; sequence predicted or annotated = 3371; no matches= 2993. These genes were annotated using Gene Ontology Consortium and the distribution of the categories in both libraries was similar. The different expression genes, selected using the fold difference (fold≥5 and ≤ 5), were 238. In order to validate the library, we tested 12 different expressed genes by real time PCR. The first six of them (SMARCC2, CDC25B, S100-8, alpha-globin, beta-globin, gamma-globin) confirmed the SAGE results. The more expressed cord blood genes were SMARCC2, CDC25B and gamma-globin. Beta-globin, and S100-8 were more expressed in bone marrow. The alpha- globin result demonstrated almost the same result between cord blood and bone marrow. CDC25B is a member of the CDC25, family of phosphatases, it activates cyclin dependent kinase CDC2 which is required for entry into mitoses. SMARCC2 is a member of SWI/SNF family proteins which display helicase and ATPase activities. It is responsible for regulation of transcription of certain genes by altering the chromatin structure around those genes. S100-8 is a calcium binding protein with a prominent role in leukocyte trafficking. It stimulates shedding of L-selectin, which is also involved in cell adhesion. It makes a role in inflammatory process towards neutrophil migration to inflammatory sites. The alpha, beta and gamma globin results were those expected based on the ontogeny. The differential gene expression here obtained could be an important tool to guide new functional studies regarding the molecular mechanisms of homing, cell cycle and cell adhesion of cord blood cells.

Transcription factors, including GATA-1 protein, play a key role in controlling the cellular proliferation and differentiation of hematopoietic stem cells. Inherited missense mutations in exon 4 of GATA-1 have been found in some families, leading to a variable degree of macrothrombocytopenia with or without abnormalities in the erythrocyte lineage. Acquired mutations in exon 2 of GATA-1 have been found in Down syndrome patients with transient myeloproliferative disorder (TMD) or megakaryoblastic leukemia (AMKL). These mutations prevent the synthesis of the full length GATA-1, but allow the synthesis of a smaller form of the protein (GATA-1s). Here, we report a novel inherited mutation, in exon 2 of the GATA-1 gene, which allows only the synthesis of GATA-1s in members of a family, who present anemia and neutropenia. A man, 19 years, was referred to our hospital due to anemia manifested at 4 years of age. Blood count showed: RBC: 3.0 x106/μl, HGB: 2.9g/dl, HCT: 8.3%, MCV: 94.4fl, MCH: 32.7pg, Retic: 0.6%, WBC: 2.6x103/μl, neutrophil: 1.0x103/μl, and PLT: 250.0x103/μl. The peripheral blood film demonstrated anisocytosis, macrocytosis, poikilocytosis, and neutrophils with pseudo Pelger-Hüet anomaly. The bone marrow aspirate and biopsy showed moderate hypocellularity with trilineage dysplasia. The presumptive diagnosis of hypocellular MDS was established. Karyoptype: 46, XY. DEB: negative. CFU-GM: 54 colonies/ml (normal range: 0.5 x104 to 1.0x104). Platelet aggregation test: 0.82 mg/ml (normal range: 0.7 to 1.2), platelet aggregation to 3μM adrenaline: absent (normal: present), and platelet aggregation to 3μM ADP: second wave absent (normal: present). The number of platelets expressing GPIX glycoprotein as well as those expressing GPIb, GPIIb, and GPIIIa were similar in patient and controls. No significant differences in the mean fluorescence indexes (MFI) for GPIX (1,214.0 vs 1,015.5), GPIb (154.0 vs 136.5), and GPIIb (100.0 vs 96.1) platelet contents were found in patient and controls. In contrast, the MFI for measurement of GPIIIa was slightly lower in patient than in controls (1,538.6 vs 1,928.1). Hypocellular MDS and similar laboratory findings were also identified in 6 additional males of the same family. Hemizygous and heterozygous 332G/C mutations in exon 2 of GATA-1 were found by sequencing PCR products in 7 males and in 4 phenotypically normal females of the family, comprising three generations. Only GATA-1s, probably resulting from alternative splicing of exon 2 was identified by RT- PCR and sequencing in affected males. In heterozygous females and normal members, both isoforms of the protein were found. These results indicate that the inherited 332G/C mutation in GATA-1 is associated with a clinical and hematological picture similar to that of MDS syndrome in contrast with the other mutations in exon 2 observed in Down syndrome, which are associated with AMKL or TMD.

The levels of HbF in sickle cell disease (SCD) can be increased by pharmacological agents such as hydroxyurea (HU), which has been shown to reduce the frequency of pain crises, hospitalizations and acute chest events requiring blood transfusions in adults with SCD. However, there is some evidence to suggest that some SCD patients show benefits from HU treatment without increase in their HbF levels. Thus, the molecular mechanism by which HU increases HbF levels and improves the clinical evolution in SCD remains unclear. This study aims to provide the global gene profile of human bone marrow of a homozygous patient three months before beginning treatment and after the initial administration of HU and to investigate groups of differentially expressed genes that could be involved in the pathways by which HU improves the clinical evolution in SCD. Using the Serial Analysis of Gene Expression (SAGE) technique, two libraries, before HU administration (HbS profile) and after HU administration (HbSHU) were performed. A total of 47.192 and 46.697 tags were analyzed for the HbS and HbSHU profiles and represented 15.735 and 15.901 distinct tags, respectively. Among these, 4.151 and 3.817 tags were no match tags that could represent new genes that remain to be identified. When both profiles were compared, 518 transcripts were determined to have statistically significant differential levels of expression (P value < 0.05). The functional annotation of transcripts, according to the Gene Ontology Consortium, showed that the categories of binding and structural molecule activity were up-regulated following HU treatment. For example, genes associated with nucleic acid binding such as Signal Transducer and Activator of Transcription 5 A, v-fos FBJ murine osteosarcoma viral oncogene homolog, Early Growth Response 1 and several ribosomal and zinc finger proteins were induced by HU treatment. Conversely, the transporter activity category was down regulated by HU treatment. Genes associated with oxygen transporter activity and other genes associated with ion binding, like S100 calcium binding protein A8 (calgranulin A) and transferrin were found to be down regulated by HU treatment. Taken together, these results strongly suggest that HU produces a significant change in the expression of bone marrow cells. Future studies of these described genes that are differentially expressed during HU treatment may contribute to further the understanding of the mechanism by which HbF acts in SCD and improves the clinical evolution of the disease. The description of new genes involved in these pathways may also represent a potential tool to identify new targets for the therapy of SCD.