Regulation Of Telomerase Reverse Transcriptase (Tert) By The Leukaemic Fusion Gene Aml1/Eto
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Date
2015-11
Authors
Moses, Emmanuel Jairaj
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Publisher
Universiti Sains Malaysia
Abstract
The chromosomal translocation t(8;21) is a common chromosomal aberration that occurs in up to about 15% of all de novo acute myeloid leukaemia (AML) cases. This translocation results in the formation of a fusion onco-protein known as AML1-ETO (also known as AML1/MTG8, RUNX1/ETO or RUNX1/RUNX1T1). Much research has been done regarding this chromosomal aberration. Patients with this chromosomal translocation tend to have better clinical prognosis. Recent studies have shown that suppression of AML/ETO caused a reduction in telomerase reverse transcriptase (TERT) levels. Nevertheless, the molecular mechanism by which this fusion onco-protein governs TERT regulation is still unknown. Therefore, the aim of this study was to investigate the role of AML/ETO in TERT regulation. It was hypothesized that AML-ETO regulates TERT via the CDKN1B (p27)/RB-E2F pathway. This hypothesis was experimentally tested using a siRNA mediated gene knockdown approach. Three different siRNA’s namely siAGF1, siCDKN1B and siSKP2 and combinations thereof were utilized. Gene knockdown experiments using siAGF1 showed that AML-ETO and TERT levels were reduced at the RNA and protein level. Other proteins that showed reductions include RB, E2F1 and SKP2. There was also an accumulation of CDKN1B (p27) protein. A G1 phase arrest in the cell cycle was observed too. Other consequential observations linked to TERT loss such as reduced clonogenicity and increased senescence were evident as well. Knockdown experiments utilizing siCDKN1B exhibited decreased levels of CDKN1B (p27) at the RNA and protein level. Levels of TERT, AML-ETO, SKP2, RB and E2F1 proteins showed marked increase. Clonogenicity of the cells were not affected and percentages of senescent cells were reduced. Interestingly, experiments with siSKP2 mirrored the knockdown effects of siAGF1 albeit to a lower extent. This includes reductions in TERT, AML-ETO and SKP2 levels, increase in CDKN1B levels, reduced clonogenicity and increased senescence. This again could be attributed to TERT loss. Overall, it is clearly shown that AML-ETO regulates TERT via the SKP2/p27/RB-E2F axis.
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Keywords
Chromosomal