Bim Promoter Is Highly Methylated in Malignant Lymphoid Cell Lines, Leading to Downregulation of Bim Expression and Protection from Apoptosis.
Bibliographic record
Abstract
Abstract Bim, a proapoptotic, BH3-only, Bcl-2 family member, is the major physiological antagonist of the antiapoptotic Bcl-2 proteins in B and T lymphocytes. It is essential for the induction of apoptosis of activated T cells following an immune response and for the homeostasis of B cells; it also plays a key role in the induction of apoptosis of early hematopoietic progenitors following cytokine-deprivation. We performed a CpG Islands prediction analysis on Bim promoter, identifying a putative CpG Island. Using a Bisulfite Modification-Clonal Sequencing Analysis (BMCSA), we investigated the methylation status of 19 CpG sites (from nucleotide −504 to +64 from the ATG start site) in the Bim promoter in 12 malignant hematological cell lines: 7 of lymphoid and 5 of myeloid origin. A minimum of 6 clones were analysed. An homogeneous, very high level of methylation was present in all the lymphoid cell lines (Average Level of Methylation (ALM) 93.4 ± 4.4% Standard Deviation [SD]) and a variable level of methylation in the myeloid cell lines (ALM 37.1 ± 32.4%). The lowest ALM was found in lymphocytes from healthy donors (15.5 ± 2.1%). Evidence of Bim promoter methylation was also found in frozen tumor samples from patients affected by NPM/ALK+ lymphomas. We treated the 12 cell lines with the demethylating agent 5-azacytidine (AZA). The changes in the methylation status of Bim promoter were evaluated by BMCSA and the corresponding induction of Bim by Real-Time PCR (TaqMan) and by Western Blot. The demethylation of Bim promoter led to a potent induction of Bim at the mRNA and protein level. In the lymphoid, NPM/ALK positive, SUDHL-1 cell line, in which a complete demethylation (from 100% to 0%) was achieved, the increase in the expression of Bim was 7.7-fold and this correlated with a potent induction of apoptosis, as assessed by TUNEL and Annexin V assays. Similar results were obtained using a different demethylating agent: 5-aza-2′deoxycytidine (DAC). To assess whether the methylation of Bim promoter is an active process, a wash-out experiment was performed on the SUDHL-1 (high level of methylation, 100%), on the PML/RAR alpha positive myeloid NB4 (intermediate level of methylation, 33%) and on the BCR/ABL positive LAMA-R cell lines (unmethylated) previously treated with AZA or DAC. This experiment showed that the demethylation is reversible and that, following remethylation, the expression of Bim at mRNA and protein level is reduced to the initial value. In the NB4 cell line, in which methylation is clustered on the last 6 CpG sites, remethylation occurs following the same pattern. No de novo methylation was seen in LAMA-R after the wash-out. To address the biological role for the methylation of Bim promoter, we generated a TET-ON inducible system for BimS (the most potent proapoptotic isoform of Bim) in the highly methylated NPM/ALK+ Karpas-299 cell line, showing that, following an induction of Bim expression, the cells are potently induced to apoptosis, as assessed by FACS using TUNEL and Annexin V assays. We conclude that Bim promoter is actively methylated in several leukemias/lymphomas of T and B origin and that its methylation is associated with the downregulation of Bim expression and with protection from apoptosis.
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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) | 0.000 | 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".