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Effects of 3-deazaadenosine on apoptosis-related gene transcripts in HL-60 cells
T Loennechen1, U Moens, H Kildalsen
1Department of Pharmacy, University of Tromsø, Norway.
Abstract:
The effect of the transmethylation inhibitor 3-deazaadenosine on transcription levels of genes associated with apoptosis was investigated in HL-60 cells. After incubation of HL-60 cells with 100 microM 3-deazaadenosine for 45 min., a schedule known to perturb transmethylation metabolites and initiate apoptosis in these cells, a 50% decrease in c-myc and a 50% increase in bcl-2 RNA steady-state levels compared to control cells were observed. Transcription levels of c-myc continued to decrease after extended exposure to 3-deazaadenosine, while bcl-2 mRNA levels dropped to 25% and 30% below those in control cells after 1.5 hr and 3 hr, respectively. The expression levels of the bcl-2 related bax gene, showed a similar pattern as bcl-2; a 60% increase was initially measured, but after 1.5 and 3 hr, bax transcripts were 80% and 70% respectively, of those found in untreated cells. Another bcl-2 related gene, bcl-x, was previously reported to generate two transcripts in human cells. The long variant bcl-x1 acts as bcl-2, while the short form bcl-xs induces apoptosis. We were unable to detect bcl-xs transcripts in untreated and 3-deazaadenosine treated cells by the highly sensitive reverse transcriptase polymerase chain reaction method. This suggests that this gene product may not be involved in 3-deazaadenosine induced apoptosis in HL-60 cells. Bcl-x1 mRNA levels, however, slowly decreased with about 50% after 1.5 or 3 hr 3-deazaadenosine treatment. It is concluded that 3-deazaadenosine initiated apoptosis affects c-myc, bcl-2, bax and bcl-x1 mRNA levels.
Insights
The transmethylation inhibitor 3-deazaadenosine alters apoptosis gene expression in HL-60 cells, decreasing c-myc and affecting bcl-2, bax, and bcl-x1 mRNA levels. This study investigates its role in apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis is a crucial cellular process regulated by various genes.
- Gene expression, particularly of apoptosis-related genes, can be modulated by small molecules.
- HL-60 cells are a human leukemia cell line frequently used to study apoptosis.
Purpose of the Study:
- To investigate the effect of the transmethylation inhibitor 3-deazaadenosine on the transcription levels of apoptosis-associated genes in HL-60 cells.
- To determine the impact of 3-deazaadenosine on the mRNA expression of c-myc, bcl-2, bax, and bcl-x genes.
- To explore the role of specific bcl-x transcripts in 3-deazaadenosine-induced apoptosis.
Main Methods:
- HL-60 cells were treated with 100 microM 3-deazaadenosine for varying durations (45 min, 1.5 hr, 3 hr).
- RNA steady-state levels of c-myc, bcl-2, bax, and bcl-x were quantified using reverse transcriptase polymerase chain reaction (RT-PCR).
- Comparison of gene expression levels between treated and untreated control cells.
Main Results:
- 3-deazaadenosine treatment led to a 50% decrease in c-myc RNA levels and a 50% increase in bcl-2 RNA levels after 45 min.
- Sustained exposure to 3-deazaadenosine resulted in decreased c-myc and bcl-2 mRNA levels.
- Expression patterns of bax and bcl-x1 mRNA were also affected, with bcl-xs transcripts undetectable.
Conclusions:
- 3-deazaadenosine significantly alters the mRNA levels of key apoptosis-related genes, including c-myc, bcl-2, bax, and bcl-x1, in HL-60 cells.
- The short variant bcl-xs transcript does not appear to be involved in 3-deazaadenosine-induced apoptosis in this cell model.
- These findings highlight the impact of transmethylation inhibition on apoptosis regulation at the transcriptional level.