Tat-immunoconjugates specifically inhibit the function of nuclear p21WAF-1/CIP-1 and differentially sensitize human breast cancer cells to antiproliferative agents

4869 p21 WAF-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 p21 WAF-1/CIP-1 to cause G 1 /S-phase arrest. Our objective in this study was to determine if these tat-anti-p21 WAF-1/CIP-1 IC’s would sensitize BC cells to antiproliferative agents that mediate their effects through p21 WAF-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-p21 WAF-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 p21 WAF-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 p21 WAF-1/CIP-1 expression 2-5-fold and caused G 1 /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-p21 WAF-1/CIP-1 IC’s. γ-radiation did not induce p21 WAF-1/CIP-1 in MDA-468 cells but growth was inhibited 31.8 ± 1.9% at 8 h post-treatment; tat-anti-p21 WAF-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 p21 WAF-1/CIP-1 associated with G 2 /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-p21 WAF-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 p21 WAF-1/CIP-1 induction and tat-anti-p21 WAF-1/CIP-1 IC’s did not increase growth inhibition. We conclude that tat-anti-p21 WAF-1/CIP-1 IC’s differentially sensitized BC cells to antiproliferative agents that mediate their effects through p21 WAF-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.