Journal of

Background and Aim: Acute promyelocytic leukemia (APL) is associated with the t(1517) ,fusing promyelocytic leukemia (PML) and retinoic acid receptor-a (RARa) genes. This disease is uniquely sensitive to treatment with all-trans retinoic acid (ATRA) and highly responsive to conventional chemotherapy. The t(1117)(q23q21) abnormality associated with a PLZF-RARa rearrangement is the commonest of the alternative translocations accounting for less than 1% APL. Blasts from PLZF-RARa cases have been found to be resistant to the differentiating effects of retinoids. In this study we aimed to determine the PLZF-RARa and fusion genes in patients with APL morphology who referred to Hematology-Oncology and BMT research center, Tehran Shariaty Hospital in 2006.

Materials & Methods: Peripheral blood and/or bone marrow samples were taken from 200 patients with APL morphology and 200 patients with other subtypes of AML. The mono-nuclear cells were enriched by centrifugation over a ficoll-isopaque gradient. RNA extracted by Trizol or TriReagent and then reverse transcribed to cDNA using random primers. PCR performed using specific primers for each fusion. PCR products electerophoresed on a 2% agarose gel containing 0.05% ethidiume bromide.

Results: In 2 (1%) patients with APL-morphology the RT-PCR analysis showed PLZF-RARa fusion transcripts.

Conclusion: It can be concluded that RT-PCR is a rapid and sensitive method for detection of abnormal fusion genes in leukemia and allows the definition of a correct strategy for treatment and subsequent minimal residual disease monitoring

Background & Aim : Chronic myeloid leukemia (CML) is a disorder of pluripotential hematopoietic stem cell that is as a myeloproliferative disease and occurs in about 15 percent of all leukemia. Two cell cycle regulatory proteins that function as tumor suppressor are P16INK4A and P14ARF. The origin of these two proteins is a human INK4A-ARF gene locus that located on chromosome 9p21. P16INK4A control retinoblastoma (Rb) and P14ARF control with p53 thought negative feedback. The purposes of this study, this was that whether these genes are preferable use as a factor in prognosis and progression of disease.

Materials and Methods: This research was a Cross sectional study.  The expression of p16INK4A and p14ARF mRNA in about 73 peripheral bloods (PB) Samples were collected from 45 CML patients at different phases of disease were assayed by reverse transcriptase polymerase chain reaction (RT-PCR). 26 samples were from patients at chronic phase before any treatment, 26 samples 3 month after treatment with imatinib, 9 samples in accelerated phase and 12 samples in Blastic phase.

Results. From 45 patients with CML, 33 patients (73%) were men and 12 patients (27%) were women. About 26 samples (35%) were p16INK4A positive and 55 samples (75%) were p14ARF RT-PCR positive. This expression of the two genes at different phases of disease were not statistically significant (p>0.05).

Conclusion: High percentage of the CML patients expressed P14ARF and P16INK4A genes. The expression of these gene at different phases of disease (diagnosis, accelerate, and Blastic phases) was not statistically significant even though, the expression of these genes was higher after the treatment.  The increased expression of these genes was probably because of the Imatinib treatment.

Background and Aim: APL is a Prevalent leukemia that Approximately included 5-10% of patients with acute myeloblastic leukemia. ATRA and recently arsenic is used for treatment. ATRA leadsto resistance to treatment and arsenic is toxic in high doses.AZT induce cell death in different ways. The purpose of this study was Assessment of effect of AZT, a telomerase inhibitor, on NB4 cell line (APL cell line) to reduce toxic effect of high dose arsenic.

Materials and Methods: In this study, viability and metabolic activityof NB4 cells, treated by different concentrations of AZT(50,100,200 µM), was assessed by trypan blue dye method and MTT assay respectively.

Results: Treated cells with AZT=50,100,200µM showed decreased viability, both in dose-dependent and time-dependent through trypan blue dye method and decreased cell metabolic activity by MTT assay.

Discussion and Conclusion: Considering that AZT is able to induce apoptosis and decrease cell activity, it seems AZT is a suitable drug for inhibiting the growth of tumor cells.

Background and Aim: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia. APL is characterized by a balanced reciprocal translocation between chromosomes 15 and 17, t(517)). Important therapeutic strategies for this disease are ATRA and Arsenic trioxide. To eliminate tumor cells with arsenic, high dose of arsenic is needed. But high dose is toxic for normal tissue. The purpose of this study is Assessment of effect of low dose of arsenic trioxide in combination with AZT on NB4 cell line (APL cell line) to reduce toxic effect of high dose arsenic.

Materials and Methods: In this study after NB4 cell line culture and proliferation, the cells treated with low dose of arsenic trioxide(0.5µM) in combination with different doses of AZT(50, 100, 200 µM) and then viability and metabolic activity was assessed by try pan blue and MTT assay respectively.

Results: Low dose of arsenic (0.5µm) alone and in combination with dose of 50µm of AZT has little effect on viability and metabolic activity but in combination with higher dose of AZT has significant effect on viability and metabolic activity and both viability and metabolic activity significantly reduced.

Conclusion: Different apoptosis- induced mechanisms cause apoptosis by arsenic and AZT. Since some of these mechanisms between AZT and arsenic are similar, so maybe these similar mechanisms cause synergic effect and significant reduction of viability and metabolic activity in combination of these two drugs.

Background and Aim: Acute leukemia is one of the main causes of cancer in the world. Now a days using natural materials as source of anticancer drugs is more recommended. HESA-A is a drug of herbal-marine origin (patented by Iranian researcher). HESA-A is composed of 50% inorganic substance، 45% organic substance (aminoenthraquinone) and 5% water. In this study effects of HESA-A، on NB4 cell line (Acute promyelocytic leukemia cells) was evaluated.

Materials and Methods: HESA-A was prepared in normal saline as a stock solution (80 mg/ml, PH=7.4), and then was sterilized. After culturing and proliferation of NB4 cell line, the cells were treated by doses of 1, 2, 4 and 8 mg/ml of HESA-A. Respectively after 72h, the percentage of viable and dead cells were counted by using Trypan blue staining in Neubanr hemocytometer. Then by MTTassay, the percentage of cell survival were determined by ELISA reader in 570nm. Finally the effects of HESA-A on apoptosis were evaluated by flocytometery.

Results: This invitro study shows that HESA-A has a cytotoxcic and antiprolifrative effects against NB4 cell line (Dose dependent).IC50 dose was 5mg/ml .HESA-A can result in apoptosis in 50% of the cells.

Conclusion: Although the mechanism of HESA-A cytotoxicity action is not known, yet this study shows that this drug may cause apoptosis of cells by dose dependent method.

Background and Aim: Chronic myelogenous leukemia is characterized by Philadelphia (Ph) chromosome, the presence of BCR-ABL fusion gene and constitutive activation of the ABL1 tyrosine kinase. Despite an excellent result of target therapy by imatinib, some patients develop resistance to imatinib. In this study Efficacy of HESA-A on proliferation and apoptosis of K562 cell line was assessed.

Materials and Methods: In this study doubling time of K562 cell line was calculated. The cells were affected by various concentrations of HESA-A(1,2,4 and 8 mg/ml respectively). Cytotoxicity and IC50 dose of HESA-A were detected by MTT and trypan blue exclusion assay. Apoptosis was assessed by flowcytometry after 48 h cell treatment in the presence of IC50 dose.

Results: Doubling time of K562 cells was 24 hours. HESA-A reduced the number of viable K562 cells in a concentration dependent manner.IC50 dose was 3.5 mg/ml. In flowcytometry analysis of apoptosis, 19.22% of the treated cells were located in the position of the necrotic cells.

Conclusion: The results of MTT and trypan blue exclusion assay suggest that HESA-A inhibits the growth of k562 cells in a concentration dependent manner and induces necrosis in K562 cells.

 Background and Aim: A goal of modern cancer research is to reach targeted therapies with drugs having fewer side effects. AZD1152 is a highly specific inhibitor of Aurora Kinase B, which leads to the programmed cell death by different mechanisms. The aim of this study was to evaluate the effects of AZD1152 on viability and metabolic activity of NB4 cells (APL derived cell line).

 Materials and Methods: The cells were treated with various concentrations of AZD1152. After 24, 48 and 72h treatments, the metabolic activity and viability of inhibitor-treated NB4 cells were assessed using MTT and trypan blue dye exclusion assays, respectively. Data were analyzed by applying student’s t-test (Microsoft Excel).

 Results: A t 25, 50 and 100 nM, AZD1152 reduced the metabolic activity by 9.2, 15.5 and 56.2% (after 24h), 10.3, 19.5 and 59.9% (after 48h), and 17.1, 28.4 and 64.8% (after 72h), respectively. Meanwhile, the percentage of viability was decreased to about 51, 45 and 40% (after 24h), 39, 36 and 30% (after 48h), and 34, 32 and 28% (after 72h), respectively.

 Conclusion : According to the results, AZD1152 has substantial efficacy on APL cell line and may be applied in some cases, e. g., for patients who have relapse or who become refractory to the conventional chemotherapy. Further studies are needed to show the molecular mechanisms regulating effects of this anti-cancer agent.

Background and Aim: FLT3 gene is a member of class III receptor Tyrosine Kinase, which is expressed in most patients with acute myeloid leukemia (AML). Mutations of FLT3 such as Internal Tandem Duplication (ITD) and point mutation of the D835 are the most common genetic defects in myeloid leukemia. These two mutations in patients with MLA and their effect on survival rate were studied for the first time in Iran.

Materials and Methods: DNA was extracted from the blood or bone marrow samples of 100 patients with AML from October 2008 to September 2009. For further analysis, PCR was performed to determine the prevalence of these mutations and their association with prognosis. Moreover, t and x² statistical tests were applied for data analysis.

Results: According to the results, 15% of patients revealed FLT3-ITD mutations and 8% showed mutation of D835 in FLT3 gene. In terms of achieving complete remission (CR), patients with mutation of ITD had more chance than those without such mutation (83.5% versus 53.3%). The white blood cell count was significantly higher in the ITD⁺ (mean = 73,646/ml) than ITD^- patients (mean = 26,580/ml).

Conclusion : The results indicate that FLT3-ITD mutations may reduce the chances of Complete Remission (CR) in patients however, FLT3-ITD mutation is an important factor in selecting appropriate treatment.

Background and Aim: Acute promyelocytic leukemia (APL) is a distinct type of leukemia which is caused due to a blockage in myeloid cells normal maturation. The most important therapeutic strategies include the use of ATRA and Arsenic trioxide. Although ATRA is generally well tolerated, some patients develop Retinoic acid syndrome. Some of the symptoms of this syndrome are directly or indirectly related to elevated WBC counts. This study aims to determine the effect of ATRA and BIBR1532 combination on WBC count as a factor leading to the formation of ATRA syndrome.

Materials and Methods: To investigate the effect of BIBR1532 and ATRA combination, NB4 cells were cultured in the presence of 30μ M and 1 μM densities of the drugs. To study the effect of drugs on living cells count, proliferation activity, and metabolic activity of the cells, Trypan blue, Brdu and MTT tests were used, respectively.

Results: The results of Trypan blue, MTT and Brdu suggest that the combination of ATRA and BIBR1532 is more effective than ATRA alone on the reduction of viable cell count, metabolic activity and proliferation of leukemic cells in the first five days of treatment.

Conclusion: The results suggest that the combination of ATRA and BIBR1532 is probably more effective in the treatment of APL patients. It seems that such improvement in results is more obvious especially among the patients who are at a higher risk of ATRA syndrome. 

Background and Aim: Indole-3-carbinol (I3C), found in Brassica species vegetables, exhibits antitumor effects. It has been shown that I3C induces apoptosis in various cell types through inactivation of the nuclear factor-kappa B (NF- k B) pathway. Anthracyclines such as doxorubicin, is widely used in the treatment of hematological malignancies, induce apoptosis in tumor cells via DNA damage and activation of p53. However, NF- k B pathway that activated by anthracyclines as a part of DNA damage response can induce chemo resistance. In this study the apoptotic effect of doxorubicin in combination with NF- k B inhibitor I3C was assessed in acute lymphoblastic leukemia cells.

Materials and Methods: Human pre-B acute lymphoblastic leukemia cell line, NALM-6 cells, were preincubated with various concentrations of I3C for1 hour and then treated with 125nM doxorubicin at 37 ° C for 24 hours. Cellular DNA content assay and Annexin V-FITC staining were performed by flowcytometry for evaluation of apoptosis.

Results: DNA histogram analysis of NALM-6 cells indicates that combination of I3C with doxorubicin synergistically escalated the percentages of sub-G1 population cells (apoptotic cells) as compared to doxorubicin-only treated group. Annexin V-FITC staining showed that cotreatment of NALM-6 cells with I3C and doxorubicin increased the proportion of Annexin-V positive cells (early apoptotic cells) in comparison with the doxorubicin treated cells.

Conclusion: The results of cell culture treatments and cell death analysis by flowcytometry suggest that I3C synergistically potentiates doxorubicin-induced apoptosis in human leukemia NALM-6 cells.

Background and Aim: Chronic lymphocytic leukemia (CLL) is a lymphoprolifrative disorder, which is very heterogeneous in prognosis. Therefore, the analysis of the prognostic factors should be very helpful in diagnosing patients with a poor prognosis. The aim of this study was to determine the relationship between circulating Endothelial cells number with VWF levels along with the other hematological findings in CLL.

Materials and Methods: Peripheral blood samples were obtained from 30 CLL patients admitted to the hematology department of Firozgar hospital and 30 healthy subjects. The levels of CEC were measured by the presence of CD34 and VWF markers, and absence of CD45 marker, using flow cytometry. VWF levels was evaluated by ELISA.

Results: The CEC levels were significantly higher in blood of the CLL patients (0.64%), when compared to the controls (0.12%, P=0.002). The levels of plasma VWF were higher among high grade patients, when compared to controls and patients in lower grades. There was a negative correlation between CEC levels, and Hb concentration in the patients group (r=-0.47, P=0.01).

Conclusion: Although the levels of both CEC count and plasma VWF in patient with high grade CLL were increased when compared to patients with low grade, there was no significant correlation between these two parameters.

Background and Aim: Cancer disease is one of the main problems of Iranian health system. It is after Cardiovascular diseases and accidents, the third leading cause of death in Iran. In many countries, differences in socio-economic status have been linked with the incidence of disease, death and in general, health inequalities. The aim of this study is to determine the socioeconomic factors associated with the incidence of leukemia in Iran.
Materials and Methods: The present descriptive-analytical study was done with panel data modeling, including information related to 30 provinces of Iran from 2004 to 2009. Socioeconomic data were collected from provincial statistical yearbooks and data on age-standardized incidence rate (ASIR) of leukemia cancer per 100,000 populations were obtained from published reports by Iran Cancer Registry. 
Results: The results showed that the leukemia incidence in men and in women during the period under review has been upward. The highest and lowest incidence of leukemia was in Yazd and Sistan provinces, respectively. Direct relationship between unemployment rate, urbanization ratio, and human development index with cancer incidence rate was evident in this study.
Conclusion: The increase of leukemia cancer in Iran has been confirmed by the current study. Leukemia cancer was significantly higher among provinces with higher socioeconomic status. This should be considered for planning support.

Background and Aim: Chronic myeloid leukemia (CML) is a myeloprolifrative neoplasm that is characterized by an expansion of myeloid, erythroid cells and platelets in peripheral blood and myeloid hyperplasia in bone marrow. Secreted frizzled-related protein family is a negative regulator of the Wnt signaling pathway that suppresses this signaling pathway in healthy individuals. Aberrant regulation of the Wnt signaling pathway is a prevalent theme in cancer biology, and methylation in promoter of SFRP family has been shown to cause uncontrolled cell proliferation in cancer. Chronic myeloid leukemia was the first malignancy in which the important role of Wnt signaling pathway has been described. 
In the present study, we examined the methylation status of SFRP1 and SFRP2 genes in patients with CML.
Materials and Methods: Blood samples were obtained from 25 healthy individuals and 33 patients whit chronic meyloied leukemia (23 male, 10 female) Then Isolated DNA was treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction (MSP) with primers specific for methylated and unmethylated promoter sequences of the SFRP1 & -2 genes. We used Mann-Whitney u-tests to investigate the correlation between SFRP-1 and SFRP-2 genes hypermethylation and clinical parameters.
Results: In CML patient hypermethyleation frequency of SFRP-1 and SFRP-2 genes were 16.1℅ and 27.2% respectively. In control group SFRP-1 and SFRP-2 genes were unmethylated.
Conclusion: The present study showed that, methylation of SFRP genes also occurs in CML like other solid tumors. Therefore, the methylation of these genes may play a role in the initiation of malignant disease.