Busulfan-induced senescence in normal lung fibroblasts.

摘要

Normal tissue damage is one of the most detrimental side effects of cancer therapy, compromising efficacy and increasing treatment duration. The induction of apoptosis in response to cancer therapy is well studied; however, senescence has emerged as am equally significant damage in the intestines, bone marrow, skin, lung, and liver. Specifically, damage to the bone marrow can lead to long term myelosuppresion, subsequent pancytopenia, and potentially secondary neoplasms. Our lab has shown that BU, a chemotherapy commonly used for treatment of CML and for myeloablation prior to bone marrow transplant, induces premature senescence and not apoptosis in hematopoietic stem cells (HSCs). The molecular mechanism remains to be elucidated, however preliminary research suggests that BU-induced senescence in HSCs is p53-independent. In order to investigate the biochemistry of BU-induced senescence, we utilized WI38 normal human lung fibroblasts; a common cell line studied in the senescence field and a cell line more conducive to cell culture and protein analysis than HSCs. For the first time to our knowledge, we show that WI38 fibroblasts incubated with BU undergo premature senescence, but not apoptosis, in a dose-independent manner. In contrast, incubation with Etoposide, a topoisomerase II inhibitor, induces WI38 cell apoptosis and/or senescence in a dose-dependent way, e.g. high dose-apoptosis and low dose-senescence. Induction of WI38 cell senescence by Etoposide is mediated via a well-established DNA damage response pathway: transient activation of the p53-p21 pathway followed by a sustained up-regulation of p16Ink4a resulting in permanent G1 arrest, whereas senescent WI38 cells induced by BU are permanently arrested in G2 phase in association with a sequential activation of the Erk (p42/44) and p38 MAPK pathways and increased expression of p16 and p21 without significant activation of p53. In addition, inhibition of Erk and p38 by specific inhibitors (PD98059 and U0126 or SB203580) significantly inhibits BU-induced senescence. Conversely, inhibition of JNK by SP600125 and p53 with alpha-PFT and siRNA has no such effect. Upon removing Erk and p38 inhibition, BU-treated cells can proceed to DNA synthesis and cell division. Additionally, we have found that BU causes an immediate, transient reduction of GSH leading to a significant, biphasic increase in ROS mediated by NAD(P)H oxidase. Inhibition of ROS with NAC prevents BU-induced activation of the MAPK cascades as well as senescence, suggesting that ROS act upstream of the Erk-p38 MAPK signaling pathway utilized{09} by BU. These findings suggest that BU is a distinctive chemotherapeutic agent that causes dose-independent senescence through a unique mechanism. A better understanding of the mechanism by which BU induces senescence could be beneficial to the effort of developing new therapeutic strategies to limit bone marrow stem cell damage and thereby improve cancer therapy efficacy.