Differential Expression of the hTERT Gene in Umbilical Cord-Derived Mesenchymal Stem Cells Cocultured with B Cell Precursor Leukemia Cell Microparticles or CD41+/CD61+ Platelet Microparticles.

Several investigations are being done to increase the short lifetime of mesenchymal stem cells (MSCs). One of the crucial genes involved in the immortalization of MSCs, hTERT (human telomerase reverse transcriptase), is activated in most publications using viral-based techniques. In this work, we investigated the use of platelet-derived (PMPs) and B cell precursor leukemia-derived microparticles as a nonviral method to trigger and compare the expression of the hTERT gene in MSCs. MSCs were extracted from the umbilical cord for the current investigation and identified using a flow cytometry approach and an inverted microscope. The Nalm-6 cell line and platelet concentrate were used to isolate microparticles (MPs). MSCs and MPs were cocultured for 14 days at 25-, 50-, and 100 µg/ml concentrations. qRT-PCR was used to research the expression of the hTERT gene. SPSS 26.0's t test was used to compare the outcomes. After coculture with platelet MPs, MSCs had higher levels of hTERT gene expression than the control group. In contrast, this gene's expression was concurrently decreased in MSCs exposed to MPs generated from Nalm-6. We demonstrated that following 14-day treatment, PMP significantly boosted the hTERT gene expression in MSCs, while the Nalm-6 MPs lowered the gene expression. However, additional studies are necessary due to the stability of hTERT gene expression and the immortalization of MSCs following exposure.

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma.

Background: The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype. Methods: The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma. Results: Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed. Conclusion: The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus.

Blood group genotyping in alloimmunized multi-transfused thalassemia patients from Iran.

OBJECTIVES: Serological methods may not be reliable for RBC antigen typing, especially in multi-transfused patients. The blood group systems provoking the most severe transfusion reactions are mainly Rh, Kell, Kidd, and Duffy. We intended to determine the genotype of these blood group system antigens among Iranian alloimmunized thalassemia patients using molecular methods and compare the results with serological phenotyping. METHODS: Two hundred patients participated in this study. Blood group phenotype and genotype were determined using the serological method and PCR-SSP, respectively. The genotypes of patients with incompatibility between phenotype and genotype were re-evaluated by RFLP-PCR and confirmed by DNA sequencing. RESULTS: Discrepancies between phenotype and genotype results were found in 132 alleles and 83 (41.5%) patients; however, there was complete accordance between the three genotyping methods. Most discrepancies were detected in Rh and Duffy systems with 47 and 45 cases, respectively, and the main discrepancy was in the FY*B/FY*B allele when serologically showed Fy(a+b+). All 39 undetermined phenotypes, due to mixed-field reactions, were resolved by molecular genotyping. CONCLUSION: Molecular genotyping is more reliable compared with the serological method, especially in multi-transfused patients. Therefore, the addition of blood group genotyping to serological assays can lead to an antigen-matched transfusion in these patients.

Developing a databank for multiple transfusion patients: Rh antigen and phenotype distribution among 3000 regular blood donors in Iran.

BACKGROUND: Rhesus (Rh) blood group system is clinically the most significant protein-based grouping system. The Rh system is of vital importance in blood transfusion, and incompatibility between the donor and recipient leads to alloimmunization. Alloimmunization is commonly seen in multiple-transfusion recipients (e.g. thalassemia patients). There are a few studies about the prevalence of Rh antigens, except for D, in Iran; and regarding the high prevalence of thalassemia in the country, in this study we have determined antigens and phenotypes of the Rh among population of regular blood donors with the aim of developing a detailed Rh databank. MATERIALS AND METHODS: This cross-sectional study randomly enrolled 3000 regular blood donors from three provinces of Sistan-Balouchestan, Khuzestan and Gilan in Iran, from September 2018 to May 2019. A fully automated system, based on hemagglutination, was used to Rh typing of blood samples. RESULTS: The prevalence of Rh antigens were as follows: D: 88.9 %; E: 30.9 %; C: 74.1 %; e: 96.2 %; and c: 72.8 %. The most common antigen and phenotype were "e" and R1r (DCcee), respectively. CONCLUSION: Due to the high rate of alloimmunization incidence against Rh blood group antigens among multiple transfusion recipients, development of regular blood donor's Rh databank can facilitate extensive matching for the Rh antigens and it likely reduces the risk of alloimmunization.

The Rh blood group system and its role in alloimmunization rate among sickle cell disease and sickle thalassemia patients in Iran.

INTRODUCTION: The alloimmunization following blood transfusion can be life-threatening. The Rh alloantibodies are one of the most common causes contributing to alloimmunization. This study aimed to evaluate the rate and causes of alloimmunization and to determine the Rh phenotypes and genotypes among sickle cell disease (SCD) and sickle thalassemia (Sß). MATERIALS AND METHODS: Our study included 104 SCD and Sß patients referring to Baghaei 2 Hospital of Ahvaz in 2019 using a non-random simple sampling method. The blood samples were collected for Rh phenotypes, alloantibody screening and identification, and molecular tests. The SSP-PCR and RFLP methods with the Pst 1 enzyme were used. RESULTS: The alloimmunization rate was 9.6% and 13.2% based on immunohematological tests and medical records, respectively. The main alloantibodies (90%) were anti-Rh, and 40% of the patients had multiple alloantibodies. A significant correlation was found between gender and alloimmunization. The phenotypes of DCce (37.5%), DCcEe (24%), Dce (20.2%), and dce (5.8%) and genotypes of R1r (25%), R1R2 (20.2%), R1R1 (18.3%), and R1R0 (10.6%) were the most prevalent. The R1R2 was a frequent genotype in Sß. CONCLUSION: R0r' and R1R0 genotypes were limited to our population in Iran. Due to the differences in RH genotypes between our population and others, the blood transfusion from other ethnicities increased our total alloimmunization rate.

RHD Genotyping of Rh-Negative and Weak D Phenotype among Blood Donors in Southeast Iran.

Background: The D antigen is a subset of Rh blood group antigens involved in the hemolytic disease of the newborn [HDFN] and hemolytic transfusion reaction [HTR]. The hybrid Rhesus box that was created after RH gene deletion, was known as a mechanism of the Rh-negative phenotype. Hybrid marker identification is used to confirm the deletion of the RHD gene and to determine zygosity. This study aims to detect this marker in Rh-negative and weak D phenotype blood donors of the southeast of Iran. Materials and Methods: The molecular analysis of the hybrid Rhesus box was performed on the 200 Rh-negative blood donors in Sistan and Baluchestan province, southeast Iran. The presence of alleles responsible for the D variants was assessed by DNA sequencing in 26 weak D phenotype donors. Results: Of the 200 Rh-negative blood samples, 198 samples were homozygous (99%), and two samples were heterozygous (1%). Heterozygous samples had RHD*01N.73 allele and the RHD*01N.18 allele. Of the 26 samples with weak D phenotype, 16 partial DLO (61%), 4 partial DBT1 (15.3%), 2 partial DV type 2 (7.7%), 1 weak D type 1, 1 weak D type 4.2.3, 1weak D type 105 and 1 RHD (S103P) (4%) were determined. Conclusion: Since RHD gene deletion is the main mechanism of the Rh-negativity in Sistan and Baluchestan provinces, a hybrid Rhesus box marker can be used in resolving RhD typing discrepancies by RHD genotyping methods.

Genotyping analysis of the MNS blood group system of thalassemia patients with alloantibodies in Iran.

BACKGROUND: Serological methods are unreliable for accurate determination of blood group antigens in multi-transfused thalassemia patients. The MNS blood group system has five high-frequency antigens. Many studies demonstrated that some antibodies including anti-S, anti-s, and anti-U may cause acute and delayed transfusion reactions and hemolytic disease of the fetus and newborn. This study aimed to determine the genotype of the MNS blood group in thalassemia patients with alloantibodies by molecular methods. MATERIAL AND METHODS: In this study, 104 blood samples from thalassemia patients were collected. The blood group phenotype for M, N, S and s antigens was determined by the tube hemagglutination method. MNS blood group genotyping was performed using PCR-SSP and DNA Sequencing methods. RESULTS: All patients were genotyped with a total of 6 pairs of primers. Discrepancies between genotype and phenotype were observed in 22 patients with S/s alleles and 2 patients with M/N alleles, however, there was full accordance between the results of SSP-PCR and DNA sequencing. The frequency of MNS blood group alleles was determined as follows: 25 % MNSs, 23 % MNss, 21 % MMSs, 9% MMSS, 9% MMss, 8% NNss, 2%MNSS, and NNSS, NNSs, MM genotypes at 1% each. CONCLUSION: In conclusion, molecular genotyping is more reliable than serological methods in multiple transfusion patients and can lead to a more compatible blood unit for transfusion in these patients.

Kidd Blood Group Genotyping for Thalassemia Patient in Iran.

We aimed to determine the JK genotype in thalassemia patients from Iran using different molecular methods to compare with phenotyping results. We also aimed to standardize for the first time, the Tetra-Primer ARMS PCR method for JK genotyping. The serology method cannot correctly determine the phenotype of blood group antigens in patients with multiple blood transfusions. Peripheral blood samples were taken from two hundred alloimmunized thalassemic patients in Tehran Adult Thalassemic Clinic. The samples were tested phenotypically by routine serological methods. After DNA Extraction, SSP-PCR was performed. DNA sequencing and PCR-RFLP were used to confirm the SSP-PCR results. Discrepancies were found between the phenotype and genotype in 32 out of 200 cases. In 16 cases phenotype was determined as Jk (a + b +) but genotype was JK*A/JK*A, in 14 cases phenotype was Jk (a + b +) while the genotype showed JK*B/JK*B, 1 case had been phenotyped as Jk (a + b -) but it was genotyped as JK*A/JK*B and 1 case had been phenotyped as Jk (a - b +) but it was genotyped as JK*A/JK*B. Serological results for a few samples could not be confirmed because of mix-field agglutination. The genotyping however verified the presence of Kidd alleles. Molecular methods are a valuable tool to predict blood group phenotypes in multi-transfused patients in order to select RBC units for a perfect matching improving blood transfusion and preventing alloimmunization. Also Tetra-Primer ARMS PCR is simple and cost effective methods that could be alternative by conventional Molecular methods.

RHD genotyping of serological weak D phenotypes in the Iranian blood donors and patients.

BACKGROUND: Most prevalent weak D types in the Caucasians molecularly defined weak D types 1, 2 or 3 and can be managed safely as RhD-positive, conserving limited supplies of RhD-negative RBCs. Therefore, identification of RHD alleles prevalence in each population could improve the policies related to accuracy of RhD typing. The aim of this study was to determine the frequency of RHD variant alleles among donors and patients for the first time in Iran. MATERIALS AND METHODS: RHD genotyping was performed on 100 blood donor and patient samples with weak D phenotype. PCR-SSP and DNA sequencing were used to identify the RHD alleles. RESULTS: Molecular analysis showed only 15 samples were RHD*weak D 1(n = 13) and RHD*weak D 3(n = 2), and no cases of RHD*weak D 2 were detected. RHD*weak 15 (n = 43) was determined as the most prevalent D variants in our population and the other weak D types follows: RHD*weak 4, 5, 80 and one case of each one: RHD*weak 8, 11, 14, 100 and 105. Partial D variants also was identified in 18 samples as follows: RHD*partial DLO, DBT1, DV2, DHK and DAU-1. CONCLUSION: The results of this study highlight the specific pattern of RHD status in the Iranian population. The weak D types 15 was the most common weak D type in the Iranian population. However, the screening for weak D types 1, 2 and 3 with 15 % frequency is also necessary for accurate RhD typing and developing clinical strategy of blood transfusion in weak D patients.

Distribution of Red Blood Cell Alloantibodies Among Transfusion-Dependent ß-Thalassemia Patients in Different Population of Iran: Effect of Ethnicity.

The best approach for prevention of alloimmunization in ß-thalassemia (ß-thal) patients is perfect matching of all red blood cell (RBC) antigens associated with clinically significant antibodies, but this is expensive and may limit the blood supply. Knowing the most common alloantibodies in transfusion-dependent ß-thal patients make it possible to establish more cost-effective matching strategies for high-risk antigens. With this in mind, we intended to determine the most common alloantibodies in different parts of Iran. A total of 480 alloimmunized ß-thal major (ß-TM) patients who were referred to the Tehran Adult Thalassemia Clinic in Tehran, Iran from all provinces between 2015 and 2017, were included in this study. Antibody screening was performed on the fresh serum of all patients. Subsequently, the specification of antibodies was identified using a panel of recognized blood group antigens. Anti-K was the most common alloantibody detected in ß-TM patients in all regions of Iran. The prevalence of this antibody reached to 37.7% in the western area, but in southeastern region, anti-E was predominant. Interestingly, the rare alloantibody anti-Kpa was detected with a high prevalence in the western region. The antibodies against E and D antigens were also encountered with high prevalence in most regions of the country. The present study demonstrated the distribution of alloantibodies in alloimmunized transfusion-dependent ß-thal patients from diverse ethnic and racial backgrounds of the Iranian population. The results of this study can be used as a basis to establish cost-effective RBC phenotyping and matching strategies for high-risk antigens in donors and chronic transfusion recipients in different regions of Iran.

Red Blood Cells are Appropriate Carrier for Coagulation Factor VIII.

AIMS: Factor VIII (FVIII) replacement therapy remains a primary treatment for hemophilia A, however, the development of FVIII antibodies (inhibitors) and short half-life of the FVIII products are the major complications. Erythrocytes may prevent rapid removal of drugs from plasma. Erythrocytes are biocompatible and non-immunogenic drug delivery. In this study, in vitro activity of FVIII encapsulated by human erythrocytes was investigated. METHODS: FVIII was loaded into erythrocytes using the hypo-osmotic dialysis technique. FVIII activity assay has been analyzed using Activated Partial Thromboplastin Time (APTT). Presence of FVIII on erythrocytes was detected by western blotting and flowcytometry using specific monoclonal antibody (abcam, U.K) against FVIII. Moreover, the osmotic fragility and hematologic parameters of FVIII-loaded carrier erythrocytes were measured. RESULTS: Our results indicated that FVIII could not cross the membrane, where plenty of FVIII was found on the surface of the carrier erythrocyte. Flow cytometery results showed that 11% of the loaded carrier erythrocytes was positive for FVIII protein on their surface. The greatest activation of FVIII in both groups including lysate and non-lysate FVIII-loaded RBCs was observed on the first day, and the coagulant activity of this factor was gradually reduced on days 3 and 5. In 1:50 dilution of both groups, significant differences in FVIII activity were observed in 1:50 dilution of both groups, especially on the 5th day. CONCLUSION: This study aims to introduce erythrocytes as appropriate carriers for FVIII to prolong the dosing intervals in the effective and safe levels for a relatively longer time.

Noninvasive Fetal Sex Determination by Real-Time PCR and TaqMan Probes.

BACKGROUND: Noninvasive fetal sex determination by analyzing Y chromosome-specific sequences is very useful in the management of cases related to sex-linked genetic diseases. The aim of this study was to establish a non-invasive fetal sex determination test using Real-Time PCR and specific probes. METHODS: The study was a prospective observational cohort study conducted from August 2018 to September 2019. Venous blood samples were collected from 25 Iranian pregnant women at weeks 7 to 25 of gestation. Cell-free DNA (cfDNA) was isolated from the plasma of samples and fetal sex was determined by SRY gene analysis using the Real-Time PCR technique. In the absence of SRY detection, the presence of fetal DNA was investigated using cfDNA treated with BstUI enzyme and PCR for the epigenetic marker RASSF1A. RESULTS: Of the total samples analyzed, 48% were male and 52% female. The RASSF1A assay performed on SRY negative cases also confirmed the presence of cell-free fetal DNA. Genotype results were in full agreement with neonate gender, and the accuracy of noninvasive fetal sex determination was 100%. CONCLUSION: Fetal sex determination using the strategy applied in this study is noninvasive and highly accurate and can be exploited in the management of sex-linked genetic diseases.

Procoagulant Activity of Red Blood Cell-Derived Microvesicles during Red Cell Storage.

BACKGROUND: Red blood cells (RBCs) undergo structural and biochemical alterations during storage which are collectively called RBC storage lesion and cause a decrease in RBC recovery and survival. During storage, erythrocytes release an increasing number of microvesicles (MVs) that have key roles in biological processes. We aimed to investigate the procoagulant activity (PCA) of RBC-derived MVs during storage. METHODS: 20 packed RBCs were stored for up to 42 days. Samples were taken at seven different times and evaluated for the presence of RBC-MVs. MVs were separated, and following filtration flow cytometry was used to characterize RBC-MVs based on the expression of glycophorin A (Gly.A) and annexin V (AnnV) antigens. The coagulant activity of RBC-MVs was tested by clotting time (CT) and PCA assays. Results were compared before and after filtration. RESULTS: Flow cytometry revealed a 17.6-fold increase in RBC-MVs after 6 weeks of storage. Significant correlations were found between AnnV+ MVs and PCA (r = 0.96; p < 0.001), and CT (r = -0.77; p < 0.001) which was associated with increased PCA and shortened CT with RBC aging. Filtration of samples efficiently removed MVs (p < 0.001) and also reduced in vitro PCA of MVs (p < 0.001). CONCLUSION: RBC-MVs are procoagulant (particularly AnnV+ MVs) Reduction of MVs from RBC concentrates may reduce the risk of transfusion-induced thrombotic complications.

microRNA expression profiles in two- and three-dimensional culture conditions of human-umbilical-cord blood-derived CD34+ cells.

Human umbilical cord blood (HUCB) is a suitable source of hematopoietic stem cells (HSCs) for therapeutic transplantation. Different approaches have been used to expand the number of HSCs to increase the rate of HSC transplantation success in patients, such as using different cocktails of cytokines, feeder cell layers, and biocompatible scaffolds. microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. They play crucial roles in hematopoiesis including stem cell proliferation, differentiation, stemness, and self-renewal properties. Here, we studied the UCB-derived CD34+ cell expansion and the miRNA signatures of CD34+ cells on two- and three-dimensional (2D and 3D) culture conditions. We successfully expanded the UCB-derived CD34+ cells in both liquid culture (2D) and on aminated polyethersulfone nanofiber scaffolds (3D). Next, we identified the miRNA signature of CD34+ cells and their target genes. We found 58 dysregulated miRNAs in 3D culture condition and 34 dysregulated miRNAs in 2D culture condition when compared to the freshly isolated CD34+ cells. Various types of target genes were also predicted in both conditions using two online databases.

RHD Genotyping by Molecular Analysis of Hybrid Rhesus box in RhD-Negative Blood Donors from Iran.

D antigen is the most important and immunogenic antigen of the Rh blood group. The RhD-negative phenotype has different genetic backgrounds with variable distribution in different populations. Hybrid Rhesus box, resulting from RHD gene deletion, is used in genotyping studies of the Rh blood group as a marker to identify the RHD gene deletion. This study for the first time identified genetic mechanisms for the occurrence of RhD-negative phenotype among the Iranian population. 200 RhD-negative blood donors were randomly selected from Tehran Blood Transfusion Center. The phenotype of D, C, Ε, e and c antigens was serologically identified, and DNA was extracted from buffy coat. The molecular analysis of hybrid Rhesus box was performed by PCR-SSP and PCR-RFLP. Moreover, the presence of different exons of RHD gene was investigated by real-time PCR on extracted DNA. Hybrid Rhesus box was detected in all samples, and PCR-RFLP confirmed that 198 (99%) were homozygous for an RHD gene deletion and 2 were heterozygous for hybrid Rhesus box in which one (0.5%) had a weak D type 11 and the other one (0.5%) had a RHD-CE (2-9)-D 2 hybrid allele. Similar to Caucasians, the frequency of RHD gene deletion was high among the Iranian population studied in this investigation, so hybrid Rhesus box can be used as an efficient marker to detect RHD gene deletion in our population.

Alteration of cellular and immune-related properties of bone marrow mesenchymal stem cells and macrophages by K562 chronic myeloid leukemia cell derived exosomes.

Leukemic cells can impact the bone marrow niche to create a tumor-favorable microenvironment using their secreted factors. Little knowledge is available about immunosuppressive and tumor-promoting properties of chronic myeloid leukemia derived exosomes in bone marrow stromal components. We report here that K562-derived exosomes can affect the gene expression, cytokine secretion, nitric oxide (NO) production, and redox potential of bone marrow mesenchymal stem cells (BM-MSCs) and macrophages. Human BM-MSCs and mouse macrophages were treated with K562-derived exosomes. Our results demonstrated that the expression of the genes involved in hematopoietic developmental pathways and immune responses, including C-X-C motif chemokine 12 (Cxcl12), Dickkopf-related protein 1 (DKK1), wnt5a, interleukin 6 (IL-6), transforming growth factor-beta, and tumor necrosis factor-alpha (TNF-alpha), changed with respect to time and exosome concentration in BM-MSCs. The TNF-alpha level was higher in exosome-treated BM-MSCs compared with the control. Exosome treatment of BM-MSCs led to an increased production of NO and a decreased production of reactive oxygen species (ROS) in a time- and concentration-dependent manner. We have shown that K562-derived exosomes induce overexpression of IL-10 and TNF-alpha and downregulation of iNOS transcript levels in macrophages. The enzyme-linked immunosorbent assay results showed that TNF-alpha and IL-10 secretions increased in macrophages. Treatment of macrophages with purified exosomes led to reduced NO and ROS levels. These results suggest that K562-derived exosomes may alter the local bone marrow niche toward a leukemia-reinforcing microenvironment. They can modulate the inflammatory molecules (TNF-alpha and NO) and the redox potential of BM-MSCs and macrophages and direct the polarization of macrophages toward tumor-associated macrophages.

Comparable osteogenic capacity of mesenchymal stem or stromal cells derived from human amnion membrane and bone marrow.

So far, substantial attentions have been attracted to the application of mesenchymal stem or stromal cells (MSCs) in different therapeutic approaches. Although human bone marrow is commonly considered as a major source for MSCs, having an invasive collection method, ethical consideration and donor availability create a challenge for scientists, leading them to explore better alternative sources for MSCs. The study presented here aimed to characterize and compare osteogenic capacity of MSCs obtained from the amnion membrane (AM) with those originated from BM. Cells isolated from AMs and BMs were cultured in DMEM-low glucose supplemented with FBS, penicillin and streptomycin. After 24 h of incubation, cells adhered to the plastic surface of the flasks were allowed to proliferate for more days. A sub-confluent culture of cells was trypsinized and re-cultured. The MSCs were characterized by the expression of specific markers with flow cytometry. The osteogenic differentiation of MSCs was also validated by alkaline phosphatase and alizarian red S staining. Our results showed comparable expression of MSCs specific markers for both MSC sources (AM and BM). We also showed the optimum osteogenic differentiation of MSCs from both sources whereas hAM-MSCs revealed higher proliferation rate. We found no essential immunophenotypic differences between MSCs originated from bone marrow and amnion membrane while their differentiations into osteoblastic linage were also comparable. This was in addition to the higher proliferation rate observed for hAM-MSCs which suggests hAM as an easily accessible and reliable source of MSCs applicable for bone engineering, regenerative medicine or other therapeutic approaches.

Harmine, a Novel DNA Methyltransferase 1 Inhibitor in the Leukemia Cell Line.

DNA methylation followed by tumor suppressor gene repression plays a critical role in the leukemia development. So, DNA methyl transferase inhibitors have great importance in treatment of theses malignancies. Harmine, A beta carboline alkaloid derivative of Peganum harmala, had shown anti- proliferative effects on leukemic cell line. This study aimed to evaluate the effect of Harmine on DNMT1 (DNA methyl transferase 1) expression in a leukemic cell line. Cell proliferation and cell cycle analysis were studied in NB4 cell line after treatment with Harmine for 72 h. DNMT1 expression in treated cells was analyzed by real time PCR. Tumor suppressor gene hypometylation and reactivation was evaluated via MSP analysis and also real time PCR. Harmine reduced cell proliferation in NB4 cell line in a time and dose-dependent manner. 102 µg/ml of Harmine was increased amount of cells in G1 Phase of cell cycle (p < 0.05). Anti proliferative doses of Harmine, has suppressed DNMT1 gene in NB4 cell line. Down-regulated DNMT1 induced p15 tumor suppressor promoter hypomethylation and reactivation. Our data indicate that Harmine can be considered as a potential treatment for AML (Acute Myeloid Leukemia), and future studies are required to test the clinical efficacy of Harmine-whether used as a single agent or as an adjuvant-for AML treatment.

Aberrant Methylation-Mediated Suppression of APAF1 in Myelodysplastic Syndrome.

Background: Myelodysplastic syndromes (MDSs) include a diverse group of clonal bone marrow disorders characterized by ineffective hematopoiesis and pancytopenia. It was found that down regulation of APAF1, a putative tumor suppressor gene (TSG), leads to resistance to chemotherapy and disease development in some cancers. In this study, we investigated the relation of APAF1 methylation status with its expression and clinicopathological factors in myelodysplastic syndrome (MDS) patients. Materials andMethods: Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) was employed in studying the methylation of CpG islands in the APAF1promoter region in MDS. Gene expression was analyzed by using real time RT-PCR. Results: 42.6% of patient samples were methylated in promoter region of APAF1analyzed, while methylation of the gene was not seen in controls (P<0.05). Methylation of APAF1was significantly associated with the suppression of its mRNA expression (P=0.00). The methylation status of APAF1in advanced-stage MDS patients (80%) was significantly higher than that of the early-stage MDS patients (28.2%) (P=0.001). The difference in frequency of hypermethylatedAPAF1 gene was significant between good (37.5%) and poor (85.71%) cytogenetic risk groups (P=0.043). In addition, a higher frequency of APAF1hypermethylation was observed in higher-risk MDS group (69.2%) compared to lower-risk MDS group (34.14%) (P=0.026). Conclusion: Our study indicated that APAF1hypermethylation in MDS was associated to high-risk disease classified according to the IPSS, WHO and cytogenetic risk.