An investigation of methylation pattern changes in the IKZF1 promoter in patients with childhood B-cell acute lymphoblastic leukemia

BACKGROUND: Ikaros family zinc finger 1 (IKZF1) is a transcription factor with an important role in controlling hematopoietic proliferation and function, particularly lymphoid cell differentiation. It was previously shown that various mechanisms and expression patterns of Ikaros are linked to a variety of cancers. We hypothesized that aberrant methylation (hypomethylation) of the IKZF1 promoter region might be one of the causes of B-cell acute lymphoblastic leukemia (B-ALL). In B-ALL patients, an increased expression of this gene is a potential cause of B-cell differentiation arrest and proliferation induction. Therefore, as more than 90% of patients with ALL are <15 years old, we investigated the methylation pattern of the IKZF1 promoter in childhood B-ALL. METHODS: Twenty-five newly diagnosed B-ALL cases were included (all younger than 15 yr). In addition, we selected 25 healthy age- and sex-matched children as the control group. We collected the blood samples in EDTA-containing tubes and isolated lymphocytes from whole blood using Ficoll 1.077 Lymphosep. Next, we extracted genomic DNA with the phenol/chloroform method. Two microgram of DNA per sample was treated with sodium bisulfite using the EpiTect Bisulfite Kit, followed by an assessment of DNA methylation by polymerase chain reaction (PCR) analysis of the bisulfite-modified genomic DNA. RESULTS: Our data highlighted a hypomethylated status of the IKZF1 promoter in the ALL cases (96% of the cases were unmethylated). In contrast, the control group samples were partially methylated (68%). CONCLUSION: This study demonstrated a hypomethylated pattern of the IKZF1 promoter region in childhood B-ALL, which might underlie the aberrant Ikaros expression patterns that were previously linked to this malignancy.

Investigation of BAX and BCL2 expression and apoptosis in a resveratrol- and prednisolone-treated human T-ALL cell line, CCRF-CEM

BACKGROUND: The numerous side effects and chemo-resistance of conventional chemical drugs in the treatment of malignancies have led to consideration of the anti-cancer properties of natural products. In the present study, we aimed to explore the apoptotic effect of two natural components, resveratrol and prednisolone, on the T acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. Our findings suggested the incorporation of these natural agents into drug regimens to treat patients with ALL. METHODS: In this study, we investigated the effect of different doses of resveratrol (15, 50 and 100 µM) and prednisolone (700 µM) on BAX (apoptosis promoter) and BCL2 (apoptosis inhibitor) expressions following 24 and 48 hours of treatment on CCRF-CEM cells, using real-time PCR, and on apoptosis induction using flow cytometry. RESULTS: The results showed a time- and dose-dependent increase in BAX expression and a decrease in BCL2 expression. Apoptosis was induced in CCRF-CEM cells treated with resveratrol and prednisolone for 24 and 48 hours. Combined resveratrol and prednisolone treatment showed synergistic effects on the overexpression of BAX and the downregulation of BCL2. The drug combination had a greater influence on apoptosis induction compared with either drug administered alone after 48 hours of treatment. CONCLUSION: The results of this study suggested that resveratrol exhibited a remarkable efficacy to improve the influence of glucocorticoids drugs, especially prednisolone, to induce apoptosis in the CCRF-CEM cell line.

Effects of hypoxia on biology of human leukemia T-cell line [MOLT-4 cells] Co-cultured with bone marrow mesenchymal stem cells

Background: One of the most significant problems in the treatment of leukemia is the expansion of resistance to chemotherapeutic agents. Therefore, assessing the drug resistance and especially the drug resistance genes of leukemic cells is important in any treatment. The impact of Mesenchymal Stem Cells [MSCs] and hypoxic condition have been observed in the biological performance of majority of leukemic cells

Methods: MOLT-4 cells were co-cultured with MSCs in the hypoxic condition induced by Cobalt Chloride [CoCl2] for 6 and 24 hr. Then, apoptosis of cells was analyzed using annexin-V/PI staining and expression of the drug resistance genes including MDR1, MRP, and BCRP along with apoptotic and anti-apoptotic genes, including BAX and BCL2, was evaluated by real-time PCR

Results: The hypoxic condition for MOLT-4 cells co-cultured with MSCs could significantly increase the expression of MDR1 and BCRP genes [p<0.05] which are involved in drug resistance. Also, the results indicated that this condition significantly increases the expression of BCL2 [p<0.05] and reduces the apoptosis in MOLT- 4 cells co-cultured with MSCs in the hypoxic condition

Conclusions: These effects can demonstrate the important role of hypoxia and MSCs on the biological behavior of Acute Lymphoblastic Leukemia [ALL] cells that may lead to particular treatment outcomes

Promoter methylation status of survival-related genes in MOLT-4 cells co-cultured with bone marrow mesenchymal stem cells under hypoxic conditions

Objective: DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells [MSCs] on the promoter methylation pattern of BAX and BCL2 genes

Materials and Methods: In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl2 was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite [SBS] treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction [MSP] was used to evaluate the methylation status of the selected genes' promoter regions

Results: The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl2and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes

Conclusion: Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and BCL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl2 induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming studies

[Effect of resveratrol on the expression of MDRl gene in MOLT-4 leukemic cell line]

The multi-drug resistance phenomena can limit the effect of chemnotherapy and lead to the recurrence of leukemia. One of the main mechanisms of multi drug resistance is the increased expression of MDRl gene that codes P-gp, a Tran's membrane carrier that exports drugs out of the leukemic cells. The aim of the study was to explore the effect of resveratrol, a natural compound, on the expression of MDRl gene in leukemic cell line MOLT-4. MTT [Methyl Tiazol Tetrazolium] assay was used to determine the sub toxic of resveratrol for treatment of leukemic cells line real Time PCR was used to determine the expression of MDRl gene. MOLT-4 cell line resistance was assessed after MTT implementation. Resveratrol could not suppress the growth of MOLT-4 cells. Genetic studies revealed increased expression of MDRl gene. Resistance of MOLT-4 cells to vincristine was increased after coadministration of vincristine and resveratrol. We did not find evidence that resveratrol can reverse multi drug resistance in MOLT4 cells

[Relationship between behavioral disorders and quality of life in patients with liver cirrhosis]

Chronic liver disease comprises a wide range of diseases characterized by inflammation of the liver and progression of cirrhosis. This study aims to assess the relationship between behavioral disorders and quality of life in patients with liver cirrhosis. The available sampling method was used to divide participants into two groups of 100 patients and 100 healthy individuals, matched for age, sex and education. Patients with cirrhosis were recruited from a subspecialty clinic in Imam Reza [AS] Hospital in Mashhad. The control group consisted of healthy participants. Participants completed the Beck Depression Inventory, State Anxiety Inventory-Trait Spielberger, Toronto Alexithymia Questionnaire and SF-36 Quality of Life Questionnaire. Research data were analyzed by Pearson correlation and multiple regression analyses. There was no significant difference between the two groups in terms of demographic variables. However significant differences existed between the variables of anxiety, depression and alexithymia. The patient group had higher depression levels compared to healthy controls. Multiple regression analysis showed that behavioral disorders, explain 30% of the variance in quality of life in patients with liver cirrhosis. These findings suggest that behavioral disorders are associated with quality of life in patients with liver cirrhosis. These results also have important implications in the field of psychopathology, prevention, and treatment of patients with cirrhosis of the liverand IV drug abuse, respectively. The prevalence of HIV coinfection amongst patients with hepatitis B or C infections was low. However, further studies with larger populations are required