Investigating the expression pattern of the angiopoietin-Tie system in ALL and its correlation with baseline characteristics

Background@#Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in children.Several environmental and genetic factors are known to be involved in its development and progression. The angiopoietin-Tie system is one of the most critical factors in angiogenesis, and its possible role in solid tumors and leukemia has been previously investigated. In this study, we examined the expression of these genes in ALL patients (early pre-B-ALL and pre-B-ALL) and compared them with normal samples. @*Methods@#Bone marrow samples were collected from 40 patients (aged 0‒19 yr) newly diagnosed with early pre-B-ALL or pre-B-ALL using molecular and flow cytometric tests and from 15 control individuals. For molecular tests, RNA extraction and cDNA synthesis were performed, and Ang1, Ang2, Ang4, Tie1, and Tie2 gene expression was examined by real-time polymerase chain reaction. @*Results@#Ang2, Tie1, and Tie2 gene expression were significantly increased in patients with ALL, whereas Ang1 gene expression was decreased. The Ang4 gene did not show significant expression changes between the two groups. @*Conclusion@#Changes in the expression of the Ang-Tie system indicate a possible role of angiogenesis in ALL prognosis. Moreover, such changes can be considered as potential diagnostic biomarkers or therapeutic targets.

Investigating the expression pattern of the angiopoietin-Tie system in ALL and its correlation with baseline characteristics

Background@#Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in children.Several environmental and genetic factors are known to be involved in its development and progression. The angiopoietin-Tie system is one of the most critical factors in angiogenesis, and its possible role in solid tumors and leukemia has been previously investigated. In this study, we examined the expression of these genes in ALL patients (early pre-B-ALL and pre-B-ALL) and compared them with normal samples. @*Methods@#Bone marrow samples were collected from 40 patients (aged 0‒19 yr) newly diagnosed with early pre-B-ALL or pre-B-ALL using molecular and flow cytometric tests and from 15 control individuals. For molecular tests, RNA extraction and cDNA synthesis were performed, and Ang1, Ang2, Ang4, Tie1, and Tie2 gene expression was examined by real-time polymerase chain reaction. @*Results@#Ang2, Tie1, and Tie2 gene expression were significantly increased in patients with ALL, whereas Ang1 gene expression was decreased. The Ang4 gene did not show significant expression changes between the two groups. @*Conclusion@#Changes in the expression of the Ang-Tie system indicate a possible role of angiogenesis in ALL prognosis. Moreover, such changes can be considered as potential diagnostic biomarkers or therapeutic targets.

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.

Different methylation patterns of RUNX2, OSX, DLX5 and BSP in osteoblastic differentiation of mesenchymal stem cells

Runt-related transcription factor 2 [RUNX2] and osterix [OSX] as two specific osteoblast transcription factors and distal-less homeobox 5 [DLX5] as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein [BSP], osteopontin and collagen type I?1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells [MSCs]. In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR [MSP]. Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction [PCR]. MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role

[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

Zoledrinic acid induces steoblastic differentiation of mesenchymal stem cells without change in hypomethylation status of osterix promoter

Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells [MSCs] has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix [OSX] promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs. In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5 micro M Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection [MOI] treatment, gene specific methylation of OSX was analyzed by methylation specific PCR [MSP]. The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation [p<0.05], but expression of OSX didn't change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards. MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases

[Epigenetic changes of RUNX2 and DLX5 genes in osteoblastic differentiation induced by zoledronic acid in mesenchymal stem cells]

Zoledronic acid as a main treatment for osteoporosis has an important role in differentiation of mesenchymal stem cells. However, mechanism of osteoblastic differentiation induction by zoledronic acid is not well understood until now. In this research we evaluate zoledronic acid effect on methylation status of RUNX2 and DLX5 promoters. After isolation and expansion of hMSCs from BM, they were pulse treated with 5 micro M ZA for 3h, and incubated in osteogenic differentiation medium for 3 weeks. DNA was extracted after first, second and third weeks of culture and also from undifferentiated MSCs. After SBS treatment, gene specific methylation analysis for RUNX2 and DLX5 were carried out by MSP using methylated and unmethylated primers. In the genes RUNX2 and DLX5, M and U primers of MSP amplified promoter regions of undifferentiated and osteoblastic differentiated MSCs. Therefore, methylation status in RUNX2 and DLX5 promoters present incomplete methylation. Methyltion patterns of RUNX2 and DLX5 don't change during zoledronic acid osteoblastic differentiation of MSCs. Our findings show that zoledronic acid does not induce osteoblastic differentiation via epigenetic mechanisms. Therefore, zoledronic acid can induce osteoblastic differentiation in a manner independent from DNA epigenetic changes

Ror2 promoter methylation change in osteoblastic differentiation of mesenchymal stem cells

Osteoblasts arise from multipotent mesenchymal stem cells [MSCs] present in the bone marrow stroma and undergo further differentiation to osteocytes or bone cells. Many factors such as proteins present in the Wnt signaling pathway affect osteoblast differentiation. ROR2 is an orphan tyrosine kinase receptor that acts as a co-receptor in the non-canonical Wnt signaling pathway. However, ROR2 has been shown to be regulated by both canonical and non-canonical Wnt signaling pathways, ROR2 expression increases during differentiation of MSCs to osteoblasts and then decreases as cells differentiate to osteocytes. On the other hand, research has shown that ROR2 changes MSC fate towards osteoblasts by inducing osteogenic transcription factor OSTERIX. Here we speculated whether ROR2 gene expression regulation during osteoblastogenesis is epigenetically determined. MSCs from bone marrow were isolated, expanded and characterized in vitro according to standard procedures. ROR2 promoter methylation status was determined using methylation specific PCR in a multipotent state and during differentiation to osteoblasts. We determined that the demethylation process in ROR2 promoter occurs during the differentiation process. The process of demethylation begins at day 8 and continues until 21 days of differentiation. This result is in concordance with previous works on the role of ROR2 on osteoblast differentiation, which have shown an upregulation of ROR2 expression during this process

Neoplastic bone marrow niche: hematopoietic and mesenchymal stem cells

The neoplastic niche comprises complex interactions between multiple cell types and molecules requiring cell-cell signaling as well as local secretion. These niches are important for both the maintenance of cancer stem cells and the induction of neoplastic cells survival and proliferation. Each niche contains a population of tumor stem cells supported by a closely associated vascular bed comprising mesenchyme- derived cells and extracellular matrix. Targeting cancer stem cells and neoplastic niche may provide new therapies to eradicate tumors. Much progress has been very recently made in the understanding of the cellular and molecular interactions in the microenvironment of neoplastic niches. This review article provides an overview of the neoplastic niches in the bone marrow. In addition to highlighting recent advances in the field, we will also discuss components of the niche and their signaling pathways