Zebularine, a cytidine deaminase and DNA methylation inhibitor, has in vitro growth inhibitory effects on human leukemia cells in association with demethylation and re-expression of p15INK4B
Bibliographic record
Abstract
1818 Promoter methylation is a major mechanism of transcriptional silencing in cancer cells that can be reversed by pharmacological treatment with known inhibitors of DNA methylation, such as 5-azacytidine (5-Aza) and its deoxy analog, 5-aza-2’-deoxycytidine (5-Aza-dC). Unfortunately, the clinical use of these drugs has been complicated by their toxicity profile and instability in aqueous solution. Recently, the cytidine analog zebularine has been proposed as an effective, stable and minimally toxic inhibitor of DNA methylation. Zebularine inhibits p16INK4A promoter methylation in T24 human bladder cancer cells in vitro and oral administration of zebularine shows in vivo antitumor effects on EJ6 human bladder cancer cells grown in mice. As promoter methylation and transcriptional silencing of the neighboring p15INK4B tumor suppressor gene occurs at a high frequency in human acute myeloid leukemia (AML), we analyzed the demethylation and anti-proliferative effects of zebularine on the human AML cell line AML193, a cell line with a methylated p15INK4B gene. Treatment of AML193 cells with zebularine resulted in a dose dependent increase in detectable p15INK4B mRNA in association with demethylation of the p15INK4B promoter CpG island. Zebularine mediated induction of p15INK4B mRNA was also associated with an increase in local acetylation of histones H3 and H4 as determined by chromatin immunoprecipitation. Interestingly, zebularine induced upregulation of p15INK4B mRNA was further augmented by co-treatment with 5-Aza-dC, a known inhibitor of DNA methylation, and the histone deacetylase inhibitor trichostatin A, with the greatest p15INK4B mRNA induction observed after treatment with all three agents. Using dosages that effectively demethylated the p15INK4B promoter, zebularine was tested for its effects on AML cell growth and was found to greatly inhibit cell viability and proliferation. Furthermore, zebularine treatment increased the percentage of cells arrested in G2/M of the cell cycle and dramatically induced apoptosis as determined by propidium iodide staining and increases in global phosphorylation of histone H3 at serine-10, histone H2A.X at serine-139, and histone H2B at serine-14. Taken together, our findings indicate that zebularine is an effective inhibitor of p15INK4B promoter methylation and cell growth in human AML in vitro. By expanding the spectrum of zebularine antitumor effects to include hematological malignancies, our study also provides further support for the clinical potential of this agent as a cancer therapy, alone, or in combination with other drugs such as 5-Aza-dC and histone deacetylase inhibitors.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".