Tat-immunoconjugates specifically inhibit the function of nuclear p21WAF-1/CIP-1 and differentially sensitize human breast cancer cells to antiproliferative agents
Bibliographic record
Abstract
4869 p21WAF-1/CIP-1 is a central downstream effector of the p53-mediated response of breast cancer (BC) cells to DNA-damaging treatments. We have designed specific HIV-1 transactivator peptide (tat) immunoconjugates (IC’s) that penetrate the membrane of BC cells and translocate to the nucleus where they inhibit the ability of p21WAF-1/CIP-1 to cause G1/S-phase arrest. Our objective in this study was to determine if these tat-anti-p21WAF-1/CIP-1 IC’s would sensitize BC cells to antiproliferative agents that mediate their effects through p21WAF-1/CIP-1. MDA-468 [high epidermal growth factor receptor (EGFR), mutant p53] and MCF-7 (low EGFR, wild type p53) were treated with camptothecin (CPT; 0.1-4 μM) or EGF (0.5-10 nM), respectively, or with γ-radiation (2-20 Gy) for 0-24 h. Treatments were given alone or combined with tat-anti-p21WAF-1/CIP-1 IC’s (5 μM). Response was evaluated at early times (24 h) after EGF or CPT exposure or γ-radiation (8 h) by studying p21WAF-1/CIP-1 induction (Western blot), cell cycle distribution (flow cytometry) and proliferation (WST-1 assay). Treatment of MDA-468 cells with EGF or CPT increased p21WAF-1/CIP-1 expression 2-5-fold and caused G1/S-phase arrest. Cell growth was inhibited 27.5 ± 0.6% or 31.8 ± 2.5% by 10 nM EGF or 1 μM CPT, respectively. Growth inhibition was significantly increased by 11.6 ± 0.4% or 29.2 ± 1.1% for EGF-treated (p=0.0001) or CPT-treated (p=0.0004) cells, respectively, when co-treated with tat-anti-p21WAF-1/CIP-1 IC’s. γ-radiation did not induce p21WAF-1/CIP-1 in MDA-468 cells but growth was inhibited 31.8 ± 1.9% at 8 h post-treatment; tat-anti-p21WAF-1/CIP-1 IC’s did not increase cell growth inhibition in these cells (26.8 ± 3.9%, p=0.311). MCF-7 cells exposed to γ-radiation or CPT had a 3-5-fold induction of p21WAF-1/CIP-1 associated with G2/M-arrest (γ-radiation) or prolonged S-phase (CPT). Cell growth was inhibited 15.4 ± 4.3% or 21.0 ± 2.5 % by 10 Gy γ-radiation at 8 h post-exposure or 1 μM CPT at 24 h post-treatment, respectively. Co-treatment with tat-anti-p21WAF-1/CIP-1 IC’s significantly increased growth inhibition by 28.9 ± 4.9% or 11.3 ± 0.9%, respectively for γ-radiation (p=0.011) or CPT (p=0.013). EGF was growth-stimulatory to MCF-7 cells; there was no effect on p21WAF-1/CIP-1 induction and tat-anti-p21WAF-1/CIP-1 IC’s did not increase growth inhibition. We conclude that tat-anti-p21WAF-1/CIP-1 IC’s differentially sensitized BC cells to antiproliferative agents that mediate their effects through p21WAF-1/CIP-1. To our knowledge, this is the first report of the use of specific tat-immunoconjugates for blocking the function of key nuclear cell cycle checkpoint proteins. This new approach provides opportunities to enhance the response to treatment in BC by interfering with protective mechanisms that rely on cell cycle arrest.
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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".