Lower Levels of TET2 Gene Expression, with a Higher Level of TET2 Promoter Methylation in Patients with AML; Evidence for the Role of Aberrant Methylation in AML Pathogenesis.

DNA methylation is a key epigenetic mechanism that is dysregulated in leukemia and plays a significant role in leukemogenesis. Ten-eleven translocation 2 (TET2) is one of the most frequently mutated genes among the DNA methylation regulators in hematologic malignancies, indicating its tumor-suppressor function. In this study, we investigated the expression and methylation status of TET2 in patients with AML. Quantitative RT-PCR was used to evaluate TET2 expression in peripheral blood mononuclear cells (PBMCs) from 51 newly diagnosed AML patients and 50 healthy controls. The methylation-sensitive high-resolution melting (MS-HRM) method was used in 45 patients with AML and 15 healthy controls to evaluate the promoter methylation of TET2. TET2 expression was significantly downregulated (P < 0.0001) in patients with AML compared to that in healthy controls. Furthermore, the methylation level of the TET2 promoter was significantly different between patients and controls. Aberrant methylation of the TET2 promoter was observed in 53.3% of the patients. Interestingly, a negative (- 0.3138) and significant (P = 0.0358) correlation between TET2 methylation and expression was found. The survival of patients with downregulated TET2 was poorer than that of other patients. TET2 gene expression was significantly downregulated while the promoter methylation was higher in patients, indicating that TET2 may be a tumor suppressor gene and a prognostic factor in AML and that transcriptional silencing of the TET2 gene may play a role in AML pathogenesis. Since epigenetic mechanisms are reversible, abnormal TET2 methylation could become a therapeutic target in the future.

Classifying tumor brain images using parallel deep learning algorithms.

One of the most important resources used in today's world is image. Medical images can play an essential role in helping diagnose diseases. Doctors and specialists use medical images to diagnose brain diseases. Convolution neural networks are among the most important deep learning methods in which several layers are trained powerfully. The proposed method is a deep parallel convolution neural network model consisting of AlexNet and VGGNet networks. The structure of the layers is different for the two types of networks. The features are combined at one point and then categorized using the softmax function. To test the network, we used different criteria with images in the database. In clinical diagnosis, generalizability means prediction for people from whom we have no observation. On this account, we did not include individuals' observations in training set in the test set. Compared to the existing models, the proposed model results show that our network has achieved better results. The best result for the database was FIGSHARE, which achieved 99.14% accuracy on binary class and 98.78% on multi-class, which was much better than other SVM models. We tested the best available result with the existing database and compared the results with the proposed model, which was the best model answer for all evaluation criteria. These results suggest that the proposed model can be used as an effective decision support tool for radiologists in medical diagnosis.

Premarital Screening Program for Congenital Factor XIII Deficiency in Iran.

BACKGROUND: Factor XIII deficiency is one of the most severe congenital bleeding disorders with high rate of life-threatening bleeding including central nervous system bleeding, umbilical cord bleeding, and recurrent miscarriages. Due to the highest global incidence of the disorder in Iran, this study aimed to design a premarital screening program in Iran. METHODS: This descriptive study was performed on 30 couples with a positive family history of factor XIII deficiency. Underling F13A gene mutation was determined in the family members, and all the selected couples underwent molecular testing mutations by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), and sequencing. RESULTS: The probability of affected childbirth existed for ten couples. Three couples decided not to marry, while seven got married and three of them decided to have a baby. The genotypes of the fetuses were determined and revealed that none of them was a homozygote for the F13A gene mutation. CONCLUSIONS: Because of the importance of factor XIII deficiency diagnosis, it can be helpful to control the incidence of factor XIII deficiency by implementing preventive programs such as premarital screening.

Polymorphisms within methotrexate pathway genes: Relationship between plasma methotrexate levels, toxicity experienced and outcome in pediatric acute lymphoblastic leukemia.

OBJECTIVES: The current study aimed to investigate the relationship of genetic polymorphism and plasma methotrexate (MTX) levels, toxicity experience and event free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: The study included 74 ALL patients. Polymerase chain reaction and genotyping of methylene tetrahydrofolate reductase (MTHFR) rs1801133, MTHFR rs1801131, ATP-binding cassette superfamily B1 (ABCB1) rs1045642, ATP-binding cassette superfamily G2 (ABCG2) rs2231142 and solute carrier 19A1 (SLC19A1) rs1051266 genetic variations were performed. The plasma MTX levels were investigated at 48 hr after the first dose of MTX infusion. RESULTS: MTHFR rs1801133 TT genotype, ABCBa1 rs1045642 CT genotype and ABCG2 rs2231142 CA genotype revealed a statistically significant association with the MTX plasma levels (P<0.01, P<0.05, P<0.05, respectively). The MTHFR rs1801133 TT genotype had a statistically significant association with hematopoietic toxicity (P<0.01) and interventions (P<0.05). The MTHFR rs1801131 AC genotype was related to the decreased hepatic toxicity (P<0.05). The SLC19A1 rs 1051266 GA genotype was related to the increased hepatic toxicity (P<0.05). Only the ABCB1 rs1045642 CT and TT genotypes had a statistically significant correlation with EFS (P<0.05, P<0.05, respectively). CONCLUSION: Our findings showed that genetic polymorphism could be associated with plasma MTX levels, toxicity experienced and EFS in Iranian pediatric ALL.

Effects of Reduced Mir-24 Expression on Plasma Methotrexate Levels, Therapy-Related Toxicities, and Patient Outcomes in Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: The current study aims to investigate the relationship of miR-24 expression with plasma methotrexate (MTX) levels, therapy-related toxicities, and event-free survival (EFS) in Iranian pediatric acute lymphoblastic leukemia (ALL) patients. METHODS: The study included 74 ALL patients in consolidation phase and 41 healthy children. RNA was extracted from plasma, polyadenylated, and reverse transcribed. miR-24 expression was determined by quantitative polymerase chain reaction (qPCR). Plasma MTX concentrations were measured by high performance liquid chromatography (HPLC) 48 h after high-dose methotrexate (HD-MTX) injection. The diagnosis of ALL was further subclassified as B-ALL or T-ALL via flow cytometry. RESULTS: miR-24 expression was less in pediatric ALL patients than in the control group (p = 0.0038). Furthermore, downregulation of miR-24 was correlated with intermediate- to high-grade HD-MTX therapy toxicities (p = 0.025). Nevertheless, no statistically significant associations were seen between miR-24 levels and plasma MTX levels 48 h after HD-MTX administration (p > 0.05) or EFS in pediatric ALL patients (p > 0.05). CONCLUSION: miR-24 expression may contribute to interindividual variability in response to intermediate- to highgrade HD-MTX therapy toxicities under Berlin Frankfurt Munster (BFM) treatment.

RITA induces apoptosis in p53-null K562 leukemia cells by inhibiting STAT5, Akt, and NF-κB signaling pathways.

Targeting oncogenic signaling pathways by small molecules has emerged as a potential treatment strategy for cancer. reactivation of p53 and induction of tumor cell apoptosis (RITA) is a promising anticancer small molecule that reactivates p53 and induces exclusive apoptosis in tumor cells. Less well appreciated was the possible effect of small molecule RITA on p53-null leukemia cells. In this study, we demonstrated that RITA has potent antileukemic properties against p53-null chronic myeloid leukemia (CML)-derived K562 cells. RITA triggered apoptosis through caspase-9 and caspase-3 activation and poly (ADP-ribose) polymerase cleavage. RITA decreased STAT5 tyrosine phosphorylation, although it did not inhibit phosphorylation of the direct BCR-ABL substrate CrkL. Real-time PCR analysis showed that RITA downregulates antiapoptotic STAT5 target genes Bcl-xL and MCL-1. The downregulation of nuclear factor-κB (NF-κB), as evidenced by inhibition of IκB-α phosphorylation and its degradation, was associated with inhibition of Akt phosphorylation in RITA-treated cells. Furthermore, consistent with the decrease of mRNA levels, protein levels of the nuclear factor-κB-regulated antiapoptotic (cIAP1, XIAP, and Bcl-2) and proliferative (c-Myc) genes were downregulated by RITA in K562 cells. In conclusion, the ability of RITA to inhibit prosurvival signaling pathways in CML cells suggests a potential application of RITA in CML therapeutic protocols.

FLT3-ITD Compared with DNMT3A R882 Mutation Is a More Powerful Independent Inferior Prognostic Factor in Adult Acute Myeloid Leukemia Patients After Allogeneic Hematopoietic Stem Cell Transplantation: A Retrospective Cohort Study

Objective: This study aimed to evaluate DNMT3A exon 23 mutations and their prognostic impacts in the presence of NPM1 and FLT3 mutations in acute myeloid leukemia (AML) patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT). Materials and Methods: This study comprised 128 adult AML patients referred to the Hematology-Oncology and Stem Cell Research Center of Shariati Hospital. NPM1 and FLT3-ITD mutations were detected by fragment analysis. For DNMT3A exon 23 mutation analysis, we used Sanger sequencing. Overall survival (OS) and relapse-free survival (RFS) curves were estimated by the Kaplan-Meier method and the log-rank test was used to calculate differences between groups. Results: The prevalence of DNMT3A exon 23 mutations was 15.6% and hotspot region R882 mutations were prominent. RFS and OS were compared in patients with and without DNMT3A exon 23 mutations using univariate analysis and there was no significant difference between these groups of patients. On the contrary, the FLT3-ITD mutation significantly reduced the OS (p=0.009) and RFS (p=0.006) in AML patients after allogeneic HSCT. In the next step, patients with AML were divided into four groups regarding FLT3-ITD and DNMT3A mutations. Patients with DNMT3A R882mut/FLT3-ITDpos had the worst OS and RFS. These results indicate that DNMT3A mutations alone do not affect the clinical outcomes of AML patients undergoing allogeneic HSCT, but when accompanied by FLT3-ITD mutations, the OS was significantly reduced (5-year OS 0% for DNMT3A R882mut/FLT3-ITDpos patients vs. 62% DNMT3A R882wt/FLT3-ITDneg, p=0.025) and the relapse rate increased. Conclusion: It can be deduced that DNMT3A R882mut/FLT3-ITDpos is an unfavorable prognostic factor in AML patients even after allogeneic HSCT.

VS-5584 as a PI3K/mTOR inhibitor enhances apoptotic effects of subtoxic dose arsenic trioxide via inhibition of NF-κB activity in B cell precursor-acute lymphoblastic leukemia.

Activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway as a survival signaling cascade is a prominent feature of cancers such as acute lymphoblastic leukemia (ALL). In patients with B-cell precursor-ALL (BCP-ALL), the high activity of the pathway correlates with the weak response to anti-leukemic drugs and relapse as a result of downstream prosurvival pathway activation, such as nuclear factor kappa B (NF-κB). Recent targeted therapy (PI3K/mTOR inhibitors) in combination with a multifunctional conventional chemotherapeutic drug may be useful for treatment of BCP-ALL patients. In the current study, the potential of a subtoxic dose (0.2 µM) of arsenic trioxide (ATO) in combination with VS-5584 (a highly potent PI3K/mTOR dual inhibitor) was tested for blocking of the PI3K/Akt/mTOR pathway, inhibition of NF-κB activation and induction of apoptosis and cell-cycle arrest. The data indicate that VS-5584 as a PI3K/mTOR inhibitor inhibited cell proliferation and induced apoptosis in NALM-6 cells by means of NF-κB transcriptional activity suppression. This apoptotic process markedly increased 72 h after administration of the subtoxic dose of ATO. We also showed that concomitant treatment of VS-5584 and the subtoxic dose of ATO significantly inhibited phosphorylation of NF-κB inhibitor alpha (IκBα) and S6 ribosomal protein (S6) as the downstream proteins of the PI3K/Akt/mTOR pathway. Combining VS-5584 and a subtoxic dose of ATO also resulted in down expression of the NF-κB target genes involved in cell proliferation and survival. These results indicate that incorporation of VS-5584/ATO combination into BCP-ALL therapeutic protocols can improve treatment and the survival of patients.

Molecular characterization of Glanzmann's thrombasthenia in Iran: identification of three novel mutations.

: Quantitative and/or qualitative defects of the platelet membrane glycoprotein IIb/IIIa complex lead to the clinical entity of Glanzmann's thrombasthenia. A large variety of mutations and polymorphisms are responsible for the aberrant expression and defective activity of this heterodimeric complex. The current study aimed to determine the pattern of mutations among Iranian population with Glanzmann's thrombasthenia. A total of 20 patients with Glanzmann's thrombasthenia have been evaluated. All exons and splice sites of ITGA2B and ITGB3 genes were amplified using touchdown PCR. Mutation screening was analyzed using conformation sensitive gel electrophoresis heteroduplex PCR, and DNA sequencing. In addition to finding one previously identified mutation and polymorphism, the experimenters explored 3 and 2 novel mutations and polymorphisms, respectively. One substitution mutation, two deletions of a single nucleotide, one insertion of a single nucleotide, two synonymous polymorphisms, and one missense polymorphism were found using Sanger sequencing method. All detected mutations were homozygous which will most likely contribute to the pathogenesis of Glanzmann's thrombasthenia. Furthermore, it suggested ITGB3 as the mainly affected gene impaired in the patients with Glanzmann's thrombasthenia. As expected, the molecular results were consistent with the phenotypic findings so that GPIIb/IIIa complex was disrupted due to mutations in all type-I Glanzmann's thrombasthenia patients. It is concluded that intronic alterations or epigenetic regulations could be responsible for aberrant expression and/or defective activity of GPIIb/IIIa complex among other patients.

Inhibition of DNA-PK enhances chemosensitivity of B-cell precursor acute lymphoblastic leukemia cells to doxorubicin.

DNA damage repair pathways greatly affect the response to genotoxic drugs in cancer cells, so inhibition of such pathways could be a potentially useful strategy to enhance chemosensitivity. DNA-dependent protein kinase (DNA-PK) plays a crucial role in the repair of DNA double-strand breaks (DSBs) that are probably one of the most detrimental types of DNA damage. It has been shown that DNA-PK is highly expressed in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Less well appreciated was the effect of DNA-PK inhibition on sensitivity of BCP-ALL cells to DNA-damaging agents. Here, we show that the DNA-PK inhibitor NU7441 increased doxorubicin-induced apoptosis in BCP-ALL cell lines (NALM-6, SUP-B15), correlating with a reduction in DSB repair measured by γ-H2AX foci. NU7441 affected the cell cycle distribution and the cell cycle regulatory molecules in combination with doxorubicin treatment. Doxorubicin-induced DNA-PK phosphorylation was decreased in the presence of NU7441. Apoptosis induction by the combined treatment was associated with marked reduction of Bcl-2 and survivin and a significant increase of Bax mRNA expression levels. In conclusion, our data indicate that inhibition of DNA-PK might be an effective approach to enhance the tumor-cell-killing effects of DNA-damaging agents such as doxorubicin in BCP-ALL and may deliver novel, targeted therapy into the clinic.

Indole-3-carbinol induces apoptosis of chronic myelogenous leukemia cells through suppression of STAT5 and Akt signaling pathways.

Signal transducer and activator of transcription 5 and Akt pathways, implicated in signaling transduction downstream of BCR-ABL, play critical roles in the pathogenesis of chronic myeloid leukemia. Therefore, idenication of novel compounds that modulate the activity of such pathways could be a new approach in the treatment of chronic myeloid leukemia. Previous studies have demonstrated that indole-3-carbinol inhibits the proliferation and induces apoptosis of various tumor cells. However, its anticancer activity against chronic myeloid leukemia cells and the underlying mechanism remain unclear. Our data revealed that indole-3-carbinol promoted mitochondrial apoptosis of chronic myeloid leukemia-derived K562 cells, as evidenced by the activation of caspases and poly (ADP-ribose) polymerase cleavage. Treatment with indole-3-carbinol was found to be associated with a decrease in the cellular levels of phospho-Akt and phospho-signal transducer and activator of transcription 5. In addition, real-time polymerase chain reaction analysis showed that the downregulation of genes is regulated by Akt and signal transducer and activator of transcription 5. We also found that treatment with indole-3-carbinol resulted in the activation of the p38 mitogen-activated protein kinase and reduced expression of human telomerase and c-Myc. Collectively, these results demonstrate that the oncogenic signal transducer and activator of transcription 5/Akt pathway is a cellular target for indole-3-carbinol in chronic myeloid leukemia cells. Thus, this clinically tested natural compound can be a potential candidate in the treatment of chronic myeloid leukemia following confirmation with clinical studies.

CREB knockdown inhibits growth and induces apoptosis in human pre-B acute lymphoblastic leukemia cells through inhibition of prosurvival signals.

A majority of acute lymphoblastic leukemia patients overexpress CREB in the bone marrow. However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of CREB on the growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells has not been elucidated. We demonstrated here that CREB knockdown induced apoptosis and impaired growth of BCP-ALL NALM-6 cells which was associated with caspase activation. The gene expression levels of prosurvival signals Bcl-2, Mcl-1, Bcl-xL, survivin and XIAP were down-regulated upon CREB suppression. These findings indicate a critical role for CREB in proliferation, survival, and apoptosis of BCP-ALL cells. The data also suggest that CREB could possibly serve as potential therapeutic target in BCP-ALL.

Laboratory Diagnosis of Factor XIII Deficiency, Routine Coagulation Tests with Quantitative and Qualitative Methods.

BACKGROUND: Factor XIII (FXIII) deficiency is a severe bleeding disorder with normal routine coagulation tests that makes diagnosis of the disorder complicated. After normal results in routine coagulation tests, clot solubility test, and FXIII activity, antigen assays along with molecular methods can be used for precise diagnosis of disorder. In the present study, we described routine coagulation tests along with clot solubility test and FXIII activity and antigen assays. METHODS: Data were collected from all relevant publications until 2015. RESULTS: All routine coagulation tests including prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), and platelet count are normal in FXIII deficiency (FXIIID) but different conditions such as blood collection, transport, and storage can result in abnormal results in these first line screening tests. In addition to these tests, clot solubility tests as the most common screening tests of FXIIID can influenced by different factors including clotting and solubilizing agents. Different commercial kits are available for FXIII activity and antigen assays with different sensitivity and specificity which could affect diagnosis of FXIIID. CONCLUSIONS: Results of routine coagulation tests as well as clot solubility tests along with specific coagulation tests can affect diagnosis of FXIIID; therefore, all steps of these tests should be under control.

Diagnosis of factor XIII deficiency.

BACKGROUND: Factor XIII (FXIII) deficiency is an extremely rare bleeding disorder with estimated incidence of one per two million. All routine coagulation tests are normal in FXIII deficiency (FXIIID), which complicates the diagnosis of this disorder. Precise diagnosis of FXIIID requires more specific tests, including qualitative tests as well as quantitative tests such as FXIII activity, antigen assays, and finally molecular studies to confirm FXIIID. OBJECTIVE: This study was conducted to present different quantitative and qualitative methods as well as molecular approaches for screening and diagnosis of FXIIID with advantages and disadvantages of each method. METHODS: All relevant English-language publications were searched in Medline (until 2015). RESULTS AND DISCUSSION: Clot solubility assay is the most widely used method for detection of FXIIID but it is not standardized. The sensitivity of this method is dependent upon different factors mainly clotting factors and the solubilizing agents; therefore, FXIII activity assay is recommended for screening of FXIIID. Among FXIII activity assays, photometric assay is more common but FXIII activity is overestimated in this assay due to lack of sample blank in commercial assay, which can have fatal consequences in severe FXIIID, for which fluorometric assay is an appropriate alternative preventing the overestimation observed in photometric assay. There are different methods for measurement of FXIII-A2, FXIII-B2, and FXIII-A2B2 as well as detection and quantification of FXIII inhibitor, which are mentioned in detail in this review. There are no mutational hotspots in FXIII-A and FXIII-B genes with a few recurrent mutations in some populations; therefore, full sequencing of FXIII genes has remained a main molecular approach for confirmation of FXIIID. CONCLUSION: Familiarity with different methods for diagnosis of FXIIID and their advantages and disadvantages can help in appropriate and timely diagnosis of this disorder to prevent misdiagnosis of FXIIID and its fatal consequences.

Disseminated intravascular coagulation with positive D-dimer: a controversial clinical feature in severe congenital factor XIII deficiency in southeast Iran.

Disseminated intravascular coagulation (DIC) is an extremely rare coagulopathy in the rare factor XIII (FXIII) deficiency. Compensated DIC occurs because of injuries that lead to systemic coagulation activation that is amplified by impaired fibrinolysis. This challenge translates into the widespread deposition of fibrin degradation products in the circulation. The aim of this study is to report three cases with severe FXIII deficiency who presented with DIC and positive D-dimer. Here we describe three patients affected by both FXIII deficiency and DIC; two girls aged 17 and 45 days and a 3.5-year-old boy. All patients had a positive family history for FXIII deficiency. Umbilical cord bleeding was the first presentation of FXIII deficiency in all of them, who presented also with ecchymosis; two of them had delayed wound bleeding. DIC occurred simultaneously with intracranial haemorrhage in two patients, whereas the third experienced DIC following extensive haematoma. D-dimer measured in all patients ranged between 5 and 20 µg/ml, whereas fibrinogen degradation product was between 4 and 8 µg/ml.

Cyclic AMP-Induced p53 Destabilization is Independent of CREB in Pre-B Acute Lymphoblastic Leukemia Cells.

Elevated cAMP levels in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells attenuate the doxorubicin-induced p53 accumulation and protect cells against apoptosis. cAMP responsive element binding protein (CREB) is a cAMP-stimulated transcription factor that regulates genes whose deregulated expression cooperate in oncogenesis. In the present study, we investigated the role of CREB on inhibitory effect of cAMP on apoptosis and p53 accumulation in BCP-ALL NALM-6 cells. To determine whether targeting CREB can modulate the effect of cAMP on doxorubicin-induced apoptosis, we knocked down CREB in NALM-6 cells using lentiviral CREB shRNA. Knocked down cells were treated with doxorubicin in the presence or absence of cAMP-elevating agents. p53 protein level and apoptosis were assessed by western blot analysis and flow cytometry, respectively. p53 protein expression was reduced in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone even in CREB-knocked down cells. Apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis in CREB-knocked down and control cells. Although, CREB plays a particularly important role in cAMP signaling pathway our data suggest that CREB does not mediate the inhibitory effect of cAMP on doxorubicin-induced apoptosis and p53 accumulation in BCP-ALL NALM-6 cells.

Elevation of cAMP Levels Inhibits Doxorubicin-Induced Apoptosis in Pre- B ALL NALM- 6 Cells Through Induction of BAD Phosphorylation and Inhibition of P53 Accumulation.

Recognition of the molecular mechanisms of cAMP action against DNA damage-induced apoptosis can be useful to improve the efficacy of DNA damaging therapeutic agents. Considering the critical role of bcl-2-associated death promoter (BAD) and p53 proteins in DNA damage -induced apoptosis, the aim of this study was to assess the effect of cAMP-elevating agents on these proteins in doxorubicin-treated pre-B acute lymphoblastic leukemia (pre-B ALL) NALM-6 cells.The pre-B ALL cell line NALM-6 was cultured and treated with doxorubicin in combination with or without cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). Cell viability was measured by trypan blue staining and MTT assay. For evaluation of apoptosis, annexin-V staining by flow cytometry and caspase-3 activity assay were used. Protein expression of p53, BAD and phoshorylated BAD was detected by western blotting analysis.cAMP-increasing agents diminished the doxorubicin-mediated cytotoxicity in NALM-6 cells as indicated by the viability assays. Annexin-V apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis. Moreover, doxorubicin-induced caspase-3 activity was attenuated in the presence of cAMP-increasing agents. Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone. Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents. However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents. Our study suggests that elevated cAMP levels inhibit doxorubicin-induced apoptosis in pre-B ALL cells through induction of BAD phosphorylation and abrogation of p53 accumulation.

MST-312 induces G2/M cell cycle arrest and apoptosis in APL cells through inhibition of telomerase activity and suppression of NF-κB pathway.

Telomerase-targeted therapy for cancer has received great attention because telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. This study was aimed to investigate the effects of telomerase inhibitor MST-312, a chemically modified derivative of epigallocatechin gallate (EGCG), on acute promyelocytic leukemia (APL) cells. Our results showed that MST-312 exerted a dose-dependent short-term cytotoxic effect on APL cells, with G2/M cell cycle arrest. Moreover, MST-312 induced apoptosis of APL cells in caspase-mediated manner. Telomeric repeat amplification protocol (TRAP) assay revealed significant reduction in telomerase activity of APL cells following short-term exposure to MST-312. Interestingly, MST-312-induced telomerase inhibition was coupled with suppression of NF-κB activity as evidenced by inhibition of IκBα phosphorylation and its degradation and decreased NF-κB DNA binding activity. In addition, gene expression analysis showed downregulation of genes regulated by NF-κB, such as antiapoptotic (survivin, Bcl-2, Mcl-1), proliferative (c-Myc), and telomerase-related (hTERT) genes. Importantly, MST-312 did not show any apoptotic effect in normal human peripheral blood mononuclear cells (PBMCs). In conclusion, our data suggest that dual inhibition of telomerase activity and NF-κB pathway by MST-312 represents a novel treatment strategy for APL.

Indole-3-carbinol suppresses NF-κB activity and stimulates the p53 pathway in pre-B acute lymphoblastic leukemia cells.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common type of cancer in children. Dramatic improvements in primary therapy for childhood ALL have led to an overall cure rate of 80 %, providing opportunities for innovative combined-modality strategies that would increase cure rates while reducing the toxic side effects of current intensive regimens. In this study, we report that indole-3-carbinol (I3C), a natural phytochemical found in cruciferous vegetables, had anti-leukemic properties in BCP-ALL NALM-6 cells. I3C induced cell growth inhibition by G1 cell cycle arrest and triggered apoptosis in a dose- and time-dependent manner. p53, p21, and Bax proteins showed increased expression after I3C treatment. Real-time PCR analysis of pro-apoptotic p53 target genes revealed up-regulation of PUMA, NOXA, and Apaf-1. I3C also suppressed constitutive nuclear factor-κB (NF-κB) activation and inhibited the protein expression of NF-kappa B-regulated antiapoptotic (IAP1, Bcl-xL, Bcl-2, XIAP) and proliferative (c-Myc) gene products. Coadministration of I3C with the topoisomerase II inhibitor, doxorubicin, potentiates cytotoxic effects compared with either agent alone. Apoptosis induction by the drug combination was associated with enhanced caspase-9 activation and PARP cleavage. Furthermore, I3C abolished doxorubicin-induced NF-κB activity as evidenced by decreased nuclear accumulation of p65, inhibition of IκBα phosphorylation and its degradation, and decreased NF-κB DNA-binding activity. Western blot analysis revealed that doxorubicin-induced Bcl-2 protein expression was inhibited by I3C. Overall, our results indicated that using nontoxic agents, such as I3C, in combination with anthracyclines might provide a new insight into the development of novel combination therapies in childhood BCP-ALL.

Association between expression of MMP-2 and MMP-9 genes and pathogenesis of intracranial hemorrhage in severe coagulation factor XIII deficiency.

Background Matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, and are involved in pathogenesis of intracranial hemorrhage (ICH). Despite replacement therapy, ICH is by far the main cause of bleeding-related death among patients with severe factor XIII deficiency (FXIIID). The aim of this study is to evaluate the association between MMP-2 and MMP-9 genes expression and ICH in these patients. Materials and methods Quantitative real-time reverse transcription (RT)-PCR assay was used to quantify MMP-2 and MMP-9 mRNAs in 42 specimens of patients with FXIIID including 18 case and 24 control groups. The comparative method of relative quantification (2-Delta-Delta-cCt) was utilized to analyze the expression level of each target gene. The expression level of glyceraldehyde-3-phosphate dehydrogenase was used to standardize the expression levels of MMPs. Results Umbilical bleeding was the most common clinical manifestation among all patients. We found expression upregulation of MMP-9 gene in 13 patients (72.2%) in case and 3 patients (12.5%) in control group, indicating a significant difference between cases and controls for MMP-9 gene expression level (P = 0.001%)(CI 2.8-95.3). Conclusions Our patients present with a wide spectrum of clinical symptoms, which are important in screening and diagnosis of patients with FXIIID. We hypothesized that the overexpression of MMP-9 due to polymorphisms or inflammation was associated with pathogenesis of ICH in FXIIID. This effect can be attenuated by nonspecific MMP inhibition to reduce hospitalization and death rates in these patients.