Abstract #1702: Molecular basis for the accelerated senescence response to doxorubicin in p53 wild-type and p53 null/mutant breast tumor cells.

Rationale: The induction of accelerated senescence by irradiation and chemotherapeutic drugs such as doxorubicin is also likely to trigger DNA damage response pathways. The generation of reactive oxygen species (ROS) has also been implicated in the induction of accelerated senescence. We evaluated the roles of several essential signaling molecules in the DNA damage response pathway as well as the involvement of ROS production during the induction of senescence by doxorubicin. Results: p53 wild type MCF-7 cells undergo widespread senescence while p53 mutant MDA-MB231 breast tumor cells and p53 null MCF-7/E6 cells show pronounced apoptosis after exposure to doxorubicin; however residual surviving MDA-MB231 and MCF-7/E6 cells demonstrated a typical senescent phenotype as well as induction of p21 after doxorubicin treatment. A luciferase reporter assay confirmed that MDA-MB231 cells do not retain any wild type p53 function. 53bp1 foci formation and up-regulation of \#947;-H2AX levels were evident in the three breast tumor cells lines, consistent with activation of the DNA damage response pathway. Inhibition of ATM activity by caffeine or KU55933 resulted in a dose-dependent blockade of doxorubicin-induced senescence in MCF-7 cells that was accompanied by a decreased induction of p21. However, suppression of two major ATM downstream effectors (Chk1 or Chk2) by UCN-01 and Chk2 inhibitor II respectively, only partially inhibited doxorubicin-induced senescence. MCF-7 cells showed a time-dependant increase of ROS generation in response to doxorubicin by DCF staining and FACS analysis. The antioxidants, glutathione (GSH) and N-acetyl cysteine (NAC), blocked doxorubicin-induced senescence in all three cell lines, suppressed doxorubicin-induced cell death, blocked the induction of p53 and p21 and down-regulation of pRb and cdc2 (all of which are associated with the accelerated senescence response to doxorubicin), and suppressed the accelerated senescence induced by the topoisomerase I poison, camptothecin. Conclusions: These studies demonstrate the central role played by ATM and p21 in accelerated senescence induced by doxorubicin and the less prominent involvement of signaling through chk1 or chk2. The basis for p53 independent induction of p21 and senescence remain to be resolved. Although the generation of reactive oxygen species contribute to accelerated senescence induced by doxorubicin (as well as camptothecin), the source of the ROS, the nature of the reactive species and their site of action (intracellular versus a membrane autocrine action) remain unclear. Similarly the basis for the tumor cell \#8220;choosing\#8221; to undergo an accelerated senescence response to stresses such as chemotherapeutic drugs that damage DNA and irradiation versus conventional growth arrest is difficult to define given the extensive overlap between these two signaling pathways.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1702.