Aylin KANLI, Hakan SAVLI, Mehmet Doğan GÜLKAÇ

Vitamin D3 (1,25(OH)2D3) ile İşlenmiş HL-60 Hücrelerindeki Gen Anlatımının Zamana Karşı Değişiminin Q-RT-PCR ile Analizi

GİRİŞ ve AMAÇ: Akut myeloid löseminin (AML) 1alpha,25- dihydroxyvitamin D (3) (1,25(OH)2D3)’ün farklılaştırma etkisiyle tedavisi ön plana çıkan araştırma konularındandır. Bu çalışmanın amacı HL-60 hücrelerinde lösemi hücre döngüsü regülasyonu ve apopitozunda 1,25(OH)2D3’ün rolünü araştırmaktır. YÖNTEM ve GEREÇLER: Vitamin D ile indüklenmiş HL-60 hücrelerinde ücre döngüsü ve apoptoz ile ilişkili 13 genin (TNFR1, Bcl-w, Bax, Bak, Caspase–6, Caspase–8, AIF, Survivin, Cdk1 (Cdc2), Cdk2, Cdk4, Siklin D1 ve Siklin E) dört farklı zamanda (18, 36, 48, 72. saatler) gen anlatımlarını gerçek zamanlı kantitatif PZR (polimeraz zincir reaksiyonu) yöntemini kullanarak analiz ettik. BULGULAR: İncelenen genlerde dramatik düşüş ve yükselişler gözlenmedi. Deneylerimizde, 72. saatte TNFR1, Cdk-4, Siklin D1, Siklin E ve Survivin genlerinin anlatımında azalma, Kaspaz-8 ve Bak genlerinin anlatımında artma gözlemledik. Hücre döngüsüne bağlı genlerin eğilimi 18. ila 72. saat arasında azalması, HL60 hücrelerinin farklılaştığını düşündürdü. Anti-apoptotik Bcl-w ve Survivin genlerinin ekspresyonunun azalması ve pro-apoptotik Bak ve Caspase-8 genlerinin ekspresyonunun artması, apoptotik işlem aşamasının 72. saatte başladığını gösterdi. TARTIŞMA ve SONUÇ: Sonuç olarak, lösemik HL60 hücre hattının D3 vitaminine maruz bırakılması hücreleri farklılaştırmıştır. Klonal gelişim tamamlanmış ve apoptotik aşama başlamıştır.

Time Dependent Gene Expression Analysis of Vitamin D3 (1,25 (OH)2 D3) Treated HL–60 Cells by Q-RT-PCR

INTRODUCTION: The treatment of Acute myelogenousleukemia (AML) with differential effect of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)2D3) has been one of thepopular research topics. The aim of this study is to investigatethe role of 1.25 (OH) 2D3 in leukemia cell cycle regulationand apoptosis in HL-60 cells.METHODS: We analyzed 13 genes (TNFR1, Bcl-w, Bax, Bak,Caspase–6, Caspase–8, AIF, Survivin, Cdk1 (Cdc2), Cdk2,Cdk4, Cyclin-D1 and Cyclin-E) for changes in expressionassociated with the cell cycle and the apoptosis of humanpromyelocytic leukemia HL-60 cells induced by 1,25(OH)2D3at 18th, 36th, 48th and 72nd h, using quantitative real-timePCR.RESULTS: We did not find significantly down or up-regulatedexpression profiles at mentioned time points. In ourexperiments, we observed decreased expression of TNFR1,Cdk-4, Cyclin-D1, Cyclin-E and Survivin genes at 72nd hours,and increased expression of Caspase-8 and Bak genes. Thetendency of cell cycle related genes decrease from 18th to 72ndhours suggested that HL60 cells differentiated. Decreasedexpression of anti-apoptotic Bcl-w and Survivin genes andincreased expression of pro-apoptotic Bak and Caspase-8genes suggested that apoptotic process stage starts around72nd hours.DISCUSSION AND CONCLUSION: In conclusion, exposureof leukemic HL60 cell line to vitamin D3 derived the cells intodifferentiation. The clonal development completed andapoptotic stage begun.

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