4 research outputs found

    Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

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    Leukemia is classified as a malignant disease of hematopoietic stem cells (HSCs) that fails in cell differentiation but preserve their self-renewal. It is caused by genetic alterations and epigenetic modifications resulting in the activation or inactivation of particular genes for transcription. Epigenetic causes changes in gene expression without any alteration in the DNA sequence. The most common epigenetic modifications are DNA methylation and histone acetylation. 5-Azacitidine (5-Aza) is a DNA methytransferase inhibitor (DNMTi) that inhibits DNA methyltransferase enzymes resulting in hypomethylation. Trichostatin A (TSA) is a histone deacetylase inhibitor which inhibits deacetylation of both histone and non-histone proteins resulting in chromatin relaxation. This present study focused on the alteration of proteome profile on 2D gel electrophoresis (2-DE) induced by 5-Aza and TSA in HL-60 and CCRF-CEM cell lines as in vitro model to represent acute promyelocytic leukemia (APL) and T-lymphoblastic leukemia (T-ALL), respectively. Total proteins of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM cell lines were extracted using urea/thiourea buffer and stained with Coomassie Blue. Comparative analysis of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM was performed by PDQuest software. Qualitative analysis identified 190-659 protein spots detected in untreated, 5-Aza and TSA-treated HL-60 and CCRF-CEM. Quantitative comparison analysis was analyzed by over 2-fold change in 5-Aza/TSA-treated cells compared to untreated. One and eight upregulated proteins were detected in 5-Aza and TSA-treated HL-60, respectively. While five and one upregulated proteins were detected in 5-Aza and TSA-treated CCRF-CEM, respectively. These preliminary results suggested that 5-Aza and TSA induced proteome profiles alterations due to their inhibition effects in HL-60 and CCRF-CEM cell lines

    Apoptotic induction in CCRF-CEM and HL-60 human leukemic cell lines by 5-Azacitidine and trichostatin A.

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    The aims of the study were to investigate the anti-cancer effects of 5Aza and TSA in two leukemic cell lines (CCRF-CEM and HL-60). Inhibition concentration of 5-Aza and TSA were measured using trypan blue exclusion assay. 5-Aza and TSA at IC50 were treated to both CCRF-CEM and HL-60 cell lines for 4-6 days. To confirm the inhibition effects of these agents, Annexin-V stained cells were analyzed using flow cytometry to evaluate the apoptotic induction. The IC50 values of CCRF-CEM were 2.01±0.1µM and 2.65±0.3µM for 5-Aza- and TSA-treated, respectively. Whereas, the IC50 values of HL-60 were 1.98±0.2µM and 2.35±0.2µM for 5-Aza- and TSA-treated, respectively. To further substantiate the findings, the time-dependent exposure of both drugs was studied. CCRF-CEM cells were reduced to 49.4%±5.0, 49.4%±2.5 and 41.5%±5.6 by 5-Aza; 56.5%±7.0, 45.3%±4.2 and 40.2%±4.2 by TSA treatment at first, third and sixth day. HL-60 cells were reduced to 72.0%±4.5, 51.0%±1.5 and 40.6%±2.6 by 5-Aza at first, third and sixth day. Meanwhile, HL-60 cells reduced to 55.6%±4.5, 45.2%±4.0 and 36.3%±2.9 by TSA at first, second and fourth day. Both cell lines were significantly inhibited (p<0.05) compared to the untreated. Furthermore, flow cytometry demonstrated that 5-Aza and TSA significantly increased the cells population positive for Annexin-V in CCRF-CEM and HL-60 cell lines. In CCRF-CEM, the total apoptotic rates were 51.7%±9.7 and 49.4%±6.0 for 5-Aza- and TSA-treated, while, in HL-60, the apoptotic rates were 51.0%±3.9 and 49.7%±9.6 for 5-Aza- and TSA-treated, in a dose- and time-dependent manner, respectively. Epigenetic modification drugs, 5-Aza and TSA have anti-leukemic effects and induce apoptosis at micro-molar concentrations in CCRF-CEM and HL-60 leukemic cell lines. These results may provide a new insight into the use of 5-Aza and TSA in inhibiting the growth of leukemic cells and useful strategy in developing an epigenetic therapy

    Table1_Molecular and hematological studies in a cohort of beta zero South East Asia deletion (β°-thal SEA) from Malaysian perspective.docx

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    AbstractWe report the haematological parameters and molecular characterization of beta zero (β°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (β°)-thalassaemia.MethodsRetrospective study on 17 cases of (β°) South East Asia (SEA) deletion from 2016 to 2019 referred to Institute for Medical Research were conducted. The clinical information and haematological profiles were evaluated. The mutation was analyzed, and the results were compared with other β°-thalassaemia groups. For HBB gene genotyping, all the cases were subjected for multiplex gap-PCR, 5 cases were subjected for HBB gene sequencing for exclusion of compound heterozygous with other beta variants. Co-inheritance of α-thalassaemia were determined using multiplex gap-PCR and multiplex ARMS-PCR.ResultsSeventeen cases were positive for β°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for β°-thal SEA deletion. The results were compared with 182 cases of various heterozygous β° deletions and mutations. The mean Hb for heterozygous β°-thal SEA deletion (13.44 ± 1.45 g/dl) was normal and significantly higher than heterozygous IVS 1-1 and Codon 41/42 (post hoc test, p ConclusionWe conclude that β°-thal SEA deletion has a unique haematological parameters of beta zero thalassaemia trait. We affirm to classifying this deletion as SEA-HPFH based on previous studies considering the phenotype features rather than the molecular defect of β°-thal SEA deletion, as this will make it easier to offer genetic counselling to affected individuals.</p

    Identification of hyaluric acid synthesis 2 (has2) and gremlin 1 (grem1) gene expressions in human cumulus cells as a biomarker for oocyte quality.

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    Nowadays, the criteria for oocyte selection is based on morphological criteria however, it needs further improvement to select the best embryo quality. The gene expression in the cumulus cell plays important role in signaling for follicular development as well as for oocyte quality. The aim of this study is to investigate the present of HAS2 and GREM1 gene expression in the cumulus cells that can become a useful marker for oocyte quality. A preliminary study was performed on cumulus cells derived from four different patients that undergo assisted reproductive technique treatment. Cumulus cells were isolated from 4 patients and the expression of HAS2 and GREM1 was analyzed by using reverse transcriptase polymerase chain reaction (rt- PCR). The results shown that HAS2 and GREM1 were expressed in grade 3 oocytes, whereas, the genes were absent in grade 4 oocytes. This showed that the expression influenced the oocyte quality. Hence, the measurement of HAS2 and GREM1 expressions in cumulus cells would reliably useful tool for selecting competence oocytes with greater chances to be fertilized in assisted reproductive technique
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