Farhad Zaker

Azidothymidine hinders arsenic trioxide-induced apoptosis in acute promyelocytic leukemia cells by induction of p21 and attenuation of G2/M arrest

To enhance anticancer efficacy of the arsenic trioxide (ATO), the combination of ATO and azidothymidine (AZT), with convergence anti-telomerase activity, were examined on acute promyelocytic leukemia (APL) cell line, NB4. In spite of an induction of apoptosis by both drugs separately and a synergistic effect of them on hTERT down-regulation and telomerase inhibition, the ATO-induced cytotoxicity was reduced when it was used in combination with AZT. AZT attenuated the ATO effects on viability, metabolic activity, DNA synthesis, and apoptosis. These observations, despite the deflection from the main goal of this study, dedicate an especial opportunity to elucidate the importance of some of the mechanisms that have been suggested by which ATO induces apoptosis. Cell cycle distribution, ROS level, and caspase-3 activation analyses suggest that AZT reduced the ATO-induced cytotoxic effect possibly via relative induction and diminution of cells accumulated in (G1, S) and (G2/M) phase, respectively, as well as through attenuation of ROS generation and subsequent caspase-3 inhibition. QRT-PCR assay revealed that induction of p21expression by the combined AZT/ATO compared to ATO alone could be a reason for the relative decline of cells accumulation in G2/M and the increase of cells in G1 and S phases. Therefore, the G2/M arrest and ROS generation are likely principle mediators for the ATO-induced apoptosis and can be used as a guide to design rational combinatorial strategies involving ATO and agents with G2/M arrest or ROS generation capacity to intensify ATO-induced apoptosis.

Direct short-term cytotoxic effects of BIBR 1532 on acute promyelocytic leukemia cells through induction of p21 coupled with downregulation of c-Myc and hTERT transcription.

Acute promyelocytic leukemia (APL) is characterized by specific t(15;17), distinct morphologic picture, and clinical coagulopathy that contribute to the morbidity and mortality of the disease. This study aims to investigate the effects of antitelomerase compound BIBR1532 on APL cells (NB4). BIBR 1532 exerts a direct short-term growth suppressive effect in a concentration-dependent manner probably through downregulation of c-Myc and hTERT expression. Our results also suggest that induction of p21 and subsequent disturbance of Bax/Bcl-2 balanced ratio as well as decreased telomerase activity may be rational mechanisms for the potent/direct short-term cytotoxicity of high doses of BIBR1532 against NB4 cells.

TGF- β1–mediated Apoptosis Associated With SMAD-dependent MitochondrialBcl-2 Expression

BACKGROUNDnTransforming growth factor (TGF) β1 can elicit various cellular responses, including inhibition of cell growth, migration, differentiation, and apoptosis. In addition, TGF-β1 is able to induce apoptosis in certain lymphomas.nnnMETHODSnIn the present study, the role of SMADs, Bax, Bcl-xl, and Bcl2 was characterized in 2 B-lymphoma cell lines, Burkitt and pre-B cell.nnnRESULTSnApoptosis was detected after exposure of TGF-β on Raji and Nalm 6 cell lines and was evaluated by flow cytometry by using annexin V, reverse transcriptase-polymerase chain reaction, and Western blot analysis. Flow Cytometry With Cell Sorting analysis showed that apoptosis could be observed after 24 hours of TGF-β treatment and was continued after 48 hours. TGF-β downregulated the Bcl-xl and Bcl-2, whereas the Bax was upregulated. Furthermore, messenger RNA of SMAD6 and SMAD7 showed the significant upregulation.nnnCONCLUSIONnThe results indicated that alteration in gene expression and protein level may determine the induction of apoptosis pathway in these lymphoma cell lines exposed to TGF-β.

FREQUENCY OF CYP1A1*2C POLYMORPHISM IN PATIENTS WITH LEUKEMIA IN THE IRANIAN POPULATION

Background: CYP1A1, a member of the cytochrome P450 (CYP) enzymes, plays a very important role in metabolizing carcinogens. The aim of this case-control study was to detect the frequency of CYP1A1*2C polymorphism in Iranian leukemic patients and determine the role of this allele’s variants, if any, as a risk factor for developing leukemia.nnMethods: Thirty-nine patients with chronic myeloid leukemia (CML), 105 with acute myeloid leukemia (AML), 95 healthy volunteers as the adult control group, 85 children with acute lymphoblastic leukemia (ALL), and 94 healthy children as the children control group were studied. Genomic DNA was assayed for restriction fragment length polymorphism (RFLP) in the CYP1A1*2C loci by amplification followed by digestion with BsrDI.nnResults: The frequencies of AA genotype (wild) were 82.05%, 62.85%, 84.70%, 85.10%, and 80% in CML, AML, ALL, the children control group, and the adult control group, respectively. The frequencies of AG genotype (heterogeneous) were 17.95%, 36.20%, 15.30%, 14.90%, and 18.95% in CML, AML, ALL, the children control group, and the adult control group, respectively. The frequencies of GG genotype (mutant) were 0.95% and 1.05% in AML and the adult control groups respectively; whereas, it was not observed in CML, ALL, or the children control group. Logistic regression analysis showed a significant correlation between the CYP1A1*2C polymorphism AG and AML patients (OR=2.4, 95% CI=1.3–4.7, P >0.05).nnConclusion: A higher frequency of CYP1A1*2C, observed in AML patients, compared with the adult control group indicates an increased risk for AML in individuals carrying the heterozygote allele CYP1A1*2C. However, the results did not show any association between CYP1A1*2C genotypes and risk of ALL or CML.nn* CYPn : cytochrome P450n CMLn : chronic myeloid leukemian AMLn : acute myeloid leukemian ALLn : acute lymphoblastic leukemian RT-PCRn : reverse transcription polymerase chain reactionn IBTOn : Iranian Blood Transfusion Organizationn EDTAn : ethylenediaminetetraacetic acidn RFLPn : restriction fragment length polymorphismn ORn : odds ration CIn : confidence interval

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Background and Objectives : Nucleophosmin gene mutations are frequently reported in acute myeloid leukemia (AML) patients with normal karyotype, which is also frequently associated with internal tandem duplication mutations in the FMS-like tyrosine kinase-3 gene. We sought to detect the nucleophosmin and FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) mutations among Iranian patients with AML and to assess the relationship between these mutations and the subtypes of the disease. Design and Setting : Cross-sectional study of patients referred during 2007 through 2009. Patients and Methods : Bone marrow and peripheral blood samples of 131 AML patients were randomly collected at the time of diagnosis and prior to treatment and the DNA extracted. After amplifying the nucleophosmin and FLT3 gene regions, positive cases were screened by conformation-sensitive gel electrophoresis and agarose gel electrophoresis techniques. Results : Of 131 patients, 23 (17.5%) (0.95% CI=0.107-0.244) had nucleophosmin gene mutations. The highest frequency of such mutations was found among the subtypes of M4 (30.4%), M3 (21.7%) and M5 (17.4%). There was a high frequency of these mutations in the M3 subtype as well as a high frequency of allele D in all subtypes. Also, 21 (16.0%) samples (0.95% CI=0.092-0.229) had FLT3/ITD mutation, of which 8 samples had mutant nucleophosmin (8 of 23, 35%), and another 13 samples had wild-type nucleophosmin gene (13 of 108, 12%). There was a high degree of association between the occurrence of nucleophosmin and FLT3/ITD mutations (P=.012). Conclusion : Our data showed a high frequency of NPM1 mutations in the monocytic subtypes of AML, as well as a high degree of association between the occurrence of NPM1 and FLT3/ITD mutations.

Evaluation of umbilical cord blood CD34+ hematopoietic stem cell expansion in co-culture with bone marrow mesenchymal stem cells in the presence of TEPA

Abstract Background During the last three decades hematopoietic stem cells (HSC) have become a standard protocol for the treatment of many hematologic malignancies and non-malignant disorders. Umbilical cord blood (UCB), as a source of HSCs, has many advantages compared with other sources. One major drawback in using this source in treatment of adult patients is the low HSC dose available. Ex vivo expansion of HSCs is a solution to overcome this limitation. In this study we used TEPA, as a Cu chelator, and human bone marrow (BM) mesenchymal stem cells (MSCs) to investigate expansion rate of UCB-HSCs. Materials and methods CB-HSCs were isolated using miniMACS magnetic separation system. We cultured the enriched CD34+cells in various conditions: culture condition A, supplemented only with recombinant cytokines; culture condition B, supplemented with BM-MSCs as a cell feeder layer and recombinant cytokines; culture condition C, supplemented with recombinant cytokines and TEPA; culture condition D, supplemented with recombinant cytokines, BM-MSCs as a cell feeder layer and TEPA. In order to evaluate the HSC expansion, we performed cell count, analysis of CD34+ expression by flow cytometry, and colony-forming cell assay on Day 10 after culture. Results The most fold increase in CD34+ cell, total cell, and total colony numbers was observed in culture condition D (110.11 ± 15.3, 118.5 ± 21, and 172.9 ± 44.7, respectively) compared to other conditions. Conclusion The results showed that co-culture of HSCs with BM-MSCs in the presence of copper chelating agent (TEPA) could dramatically increase expansion rate of UCB-HSCs. Therefore, this strategy could be useful for HSC expansion.

BIBR 1532 Increases Arsenic Trioxide-mediated Apoptosis in Acute Promyelocytic Leukemia Cells: Therapeutic Potential for APL

The current treatment of acute promyelocytic leukemia with arsenic trioxide (ATO) has increased long-lasting complete remissions; however, a proportion of patients continues to die eventually as a result of disease recurrence. In an effort to enhance the effectiveness of the APL treatment, we designed experiments to evaluate the effects of ATO in combination with the lead compound of non-nucleoside inhibitor of telomerase, BIBR 1532. After combined treatments with BIBR 1532 and ATO, decreased cell viability index with a concomitant increase in apoptotic cell death was observed in NB4 leukemic cells. Apoptosis induced by the combined treatments was accompanied by elevated Bax/Bcl-2 molecular ratio and enhanced caspase 3 activation. Our study has also demonstrated that the combined treatment suppressed NB4 cell proliferative capacity and inhibited telomerase activity probably via transcriptional suppression of c-Myc and hTERT. In conclusion, this study may supply insight into the application of this new combination therapy to APL cells intrinsically less sensitive to routine therapies and suggested a novel combination therapy for patients with more aggressive disease; those who may not respond favorably to the arsenic mono-therapy.

Cyclic AMP-induced p53 destabilization is independent of EPAC in pre-B acute lymphoblastic leukemia cells in vitro.

Context: Activation of the tumor suppressor protein p53 facilitates the cellular response to genotoxic stress. Thus, releasing the wild-type p53 from indirect suppression would be crucial to successful killing of cancer cells by DNA-damaging therapeutic agents. Objective: The aim of this study was to investigate the inhibitory role of cyclic adenosine monophosphate (cAMP) levels on p53 protein in acute lymphoblastic leukemia (ALL) cells. More importantly, we were interested to show through which receptor cAMP acts to promote p53 degradation. Materials and methods: In cell cultures, we investigated the effects of forskolin/3-isobutyl-1-methylxanthine (IBMX) on stimulated p53 of ALL cell lines. Western blotting analysis was performed to detect the expression of p53, phospho-p53, acetylated-p53, phospho-cAMP response element-binding protein (CREB), and Mdm2 proteins. Flow cytometry was applied to analyze apoptosis. The gene expression of p53 and its target genes was examined by real-time polymerase chain reaction. Results: We show that elevation of cAMP levels in ALL cells exposed to DNA damage attenuates p53 accumulation. Inhibition of proteosome function with MG-132 reversed the inhibitory effect of cAMP on p53. However, targeting the p53–Mdm2 interaction did not rescue accumulated p53 from the destabilizing signal of cAMP. The specific agonist of the cAMP receptor exchange protein activated by cAMP had no effect on p53 expression in doxorubicin-treated NALM-6 cells, whereas PKA activators decreased p53 accumulation. Discussion and conclusion: Our studies demonstrate that cAMP-PKA pathway regulates the sensitivity toward DNA-damaging agents via inhibition of a p53-dependent pathway in B-cell precursor ALL (BCP-ALL) cells.

Evaluation of T315I mutation frequency in chronic myeloid leukemia patients after imatinib resistance

Abstract The occurrence of resistance mutations in the Abl kinase domain plays a central role in drug treatment failure in chronic myeloid leukemia (CML) patients. Among them, the T315I mutation at the gatekeeper position affects a common Abl kinase contact residue and confers complete resistance to all known ATP-competitive BCR-ABL inhibitors. In the present study, an allele-specific oligonucleotide reverse transcriptase polymerase chain reaction assay was used to detect T315I mutation in a cohort of 60 imatinib-resistant CML patients. In terms of disease phase, 43 patients (71%) were in late chronic phase, 4 (7%) in accelerated phase, and 13 (22%) in blastic phase. The prevalence of the T315I mutation was found to be 7% (4/60). All four patients with mutation were in advance phases and had previously lost all their responses. The results of the study confirmed that this method is low cost and easy tool to operate for T315I mutation screening and direct sequencing should be performed in positive cases for confirmation.

Polymorphisms of the methylene tetrahydrofolate reductase and susceptibility to acute lymphoblastic leukemia in children

Background: Correlation between epigenetic factors and their effects on hematopoietic cells has led to a study of 2 common functional polymorphisms (C677T and A1298C) of 5,10methylene tetrahydrofolate reductase (MTHFR) enzyme. The aim of this study was to assess the individual and/or combined roles of these 2 polymorphisms in pediatric acute lymphoblastic leukemia (ALL). Methods: Using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses, we studied the frequencies of the C677T and A1298C MTHFR genotypes in 103 pediatric ALL patients and 160 age-sex matched controls. We calculated the odds ratio (OR) of MTHFR genotypes to determine if 1 or both of these polymorphisms may be associated with childhood ALL. Results: The T allele frequency for MTHFR 677C>T was 22.2% and 18.45% in controls and cases, respectively. The C allele frequency for MTHFR 1298 A>C was 40.65% and 40.72% in controls and cases, respectively. The OR for MTHFR 677CT was 1.08 (95%CI 0.58-1.95) and OR for MTHFR 677TT was 0.25 (95%CI 0.05-10.24). The OR for MTHFR 1298 AC was 0.57 (0.95% CI 0.57-1.95) and for MTHFR CC was 0.96 (0.95% CI 0.37-2.45). The OR for the combined heterozygous status (677CT and 1298AC) was 1.08 (95% CI 0.41-2.82). Conclusion: Our findings suggest that the MTHFR C677T and A1298C gene variants lack a major influence on the susceptibility for pediatric ALL. Another result was that the C allele frequency for MTHFR 1298 A>C was significantly higher than those reported for most Asian and European populations. The C677T prevalence seems to be similar to those reported in most Asian populations.