Abstract 2511: Differential cell cycle-dependent Chk1 inhibitor sensitization of genotoxic agents
Bibliographic record
Abstract
Abstract Chk1 kinase is a key component of the DNA damage checkpoint pathway and inhibitors have been developed to sensitize cell killing by genotoxic agents. UCN-01 and related Chk1 inhibitors abrogate DNA damage checkpoint arrest that results in mitotic catastrophe. We have characterized the mitotic defects of cells that were treated with genotoxic agents alone and in combination with Chk1 inhibition. Gemcitabine is used to treat pancreatic cancer and studies have shown that cell killing can be enhanced by Chk1 inhibitors. PANC-1 cells expressing GFP:H2B were monitored by FACs and time-lapse microscopy after treatment with gemcitabine and UCN-01. Gemcitabine induced an S phase arrest for as long as 48h. Addition of UCN-01 to S phase arrested cells promoted entry into mitosis. The morphology of the chromosomes was highly abnormal and cells were delayed in mitosis for many hours. Staining for centromere (ACA) and kinetochore (CENP-F) markers revealed that these structures appeared to be detached from the bulk of the chromatin mass, which tended to be excluded outside of the bipolar spindle. Electron microscopy of gemcitabine + UCN-01 treated cells revealed unpaired kinetochore fragments that were detached from the chromosome. These structures were reminiscent of those seen in rodent CHO cells that underwent ‘mitosis with unreplicated genomes’ (MUGs). Analysis of additional pancreatic tumor cell lines for their responses to gemcitabine + UCN-01 showed that MiaPaCaII cells MUGged but others (CFPAC and BXPC3) did not. We next tested whether insensitivity of the non-responders to UCN-01 was due to limiting factors required for the S/G2 transition or the G2/M transition. CFPAC and BXPC3 cells that were arrested in G2 with MMS or topoII inhibitors were able to enter mitosis after UCN-01 treatment. For these cells, inhibition of Chk1 is not sufficient to overcome the gemcitabine-induced S phase arrest. We discovered that kinetochore fragmentation (MUGging) resulting from Chk1 inhibition is not restricted to replication inhibitors such as gemcitabine. Intriguingly, Chk1 override of topoisomerase II inhibitor-induced G2 arrest generated MUG's in all cell lines tested, regardless of their response to gemcitabine. EM studies show the presence of detached kinetochore fragments consistent with the MUG's observed after gemcitabine+UCN-01 treatment. Our findings suggest that centromere and kinetochore fragmentation is a major mechanism by which Chk1 inhibitors sensitize cells to killing by genotoxic agents. Detachment of centromeres from chromosomes results in death of cells while arrested in mitosis. Cells that exit mitosis are multinucleated and are not clonogenically viable. Due to the sensitization of cell death when cells are forced to MUG, we propose that inhibiting Chk1 after chemotherapies that induce centromere defects such as DNA replication or DNA topology inhibitors represents a beneficial therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2511.
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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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.005 | 0.001 |
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".