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Fulltext Apoptotic induction in cCCRF-CEM and HL human leukemic cell lines by 5-azacitidine and trichostatin A

Aziee, S., Haiyuni, MY, Shafini, MY, Johan, MF, Al-Jamal, HAN, Abdul Wahab, R., et al.

Journal of Biomedical and Clinical Sciences, 2018;3(1):54-61.
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The aims of the study were to investigate the anti-cancer effects of 5-
Aza 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
Fulltext Isolation of nucleated red blood cell from peripheral blood of β-Thalassemia major patients using CD71 magnetic beads and future application

Haiyuni, M.Y., Aziee S., Heba A., Rosline H., Abdullah W.Z., Johan M.F., et al.

Journal of Biomedical and Clinical Sciences, 2018;3(1):25-30.
MyJurnal

Introduction: Isolation of specific cell types is important in providing a better understanding of hematological disorders. The knowledge of molecular biology aspect in β-thalassemia is still limited. This is because hemoglobin disorder involves various erythropoietic processes in which the genetic information is lack due to enucleation of red blood cells occurs in bone marrow. It is invasive to collect samples from bone marrow and cord blood although nucleated red blood cells (NRBCs) are abundant in these sites. NRBCs are precursors of red blood cells and typically found in peripheral blood (PB) of β-thalassemia major patients and abundant post-splenectomy. The utilization of PB NRBCs will provide a further understanding of the molecular aspects of ineffective erythropoiesis in β-thalassemia major patients. Objective: The objective of this study was to isolate the NRBCs using CD71 magnetic beads from PB of β-thalassemia major; non-splenectomy and post-splenectomy patients. Methods: NRBCs were isolated from 6 mL PB of β-thalassemia major patients based on density gradient and magnetic activated cell sorting (MACS) for NRBCs enrichment using a CD71 marker. Cell count was determined by using hemocytometer (Weber Scientific, NJ, USA) and BD FACSCantoTM II flow cytometry (Becton-Dickson, NJ, USA) was performed for method validation. Results: NRBCs were successfully isolated from the PB of both non-splenectomy and post-splenectomy β-thalassemia major patients with >90% specificity by flow cytometric analysis. The median number of enriched NRBCs (x104 ) was 58.5 (283) and 340 (338) respectively using hemocytometer. Conclusion: The MACS method was found to be convenient and efficient in the isolation of the targeted cells for downstream applications.
Conformation sensitive gel electrophoresis for the detection of calreticulin mutations in BCR-ABL1-negative myeloproliferative neoplasms

Zakaria NA, Rosle NA, Siti Asmaa MJ, Aziee S, Haiyuni MY, Samat NA, et al.

Int J Lab Hematol, 2021 Dec;43(6):1451-1457.
PMID: 34125992 DOI: 10.1111/ijlh.13628

INTRODUCTION: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients.
METHODS: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing.
RESULTS: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2.
CONCLUSION: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

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