Abstract 4671: Assessment of molecular processes relative to DNA damage response using primary human hematopoietic cells exposed to high-dose chemotherapy

Abstract

Abstract A major adverse side effect in many cancer treatments is irreversible damage to primitive and mature hematopoietic cells. Depending of the level and duration of therapy, this can subsequently lead to myelosuppression or life-threatening myeloablation. Understanding molecular processes that regulate how hematopoietic cells respond to and repair chemotherapy-mediated DNA damage could provide key information towards development of cancer treatments with acceptable hematotoxicity. However, model systems designed to study the response of human hematopoietic cells to cytotoxic therapy are lacking. An in vitro culture system that allows for robust expansion of primary human hematopoietic cells was developed. Primary human CD34+ cells were incubated in granulocyte-colony stimulating factor and stem-cell factor for 10 days. This yielded large numbers of human myeloid CD33+ cells so that activation of DNA damage response mechanisms induced by high-dose chemotherapy could be interrogated. A combination therapy of O6-benzylguanine (O6BG) and temozolomide (TMZ)-a regimen previously shown to cause myelosuppression in clinical trials-was used. Exposure of myeloid cells to O6BG/TMZ led to expected decreases in O6-methylguanine DNA methyltransferase and increases in p53, p21, H2AX, and cell cycle arrest. TMZ exposure also led to increased mitochondrial DNA damage which was further pronounced when MGMT activity was inhibited pharmacologically by O6BG, suggesting that MGMT may play a role in repairing mitochondrial DNA damage in human myeloid cells. The extrinsic versus intrinsic apoptotic pathways were also analyzed to determine if a predominant mode of cell death was evident in the myeloid cells following exposure to O6BG/TMZ. Increases in mitochondrial membrane depolarization as well as caspase-9 and caspase-3 activities were observed in cells treated with O6BG/TMZ. Additionally, while ∼50% of the myeloid cells expressed the Fas receptor, expression increased to 80-100% following exposure to 6BG/TMZ. Therefore, to what extent Fas-mediated signaling was operative following 6BG/TMZ exposure was investigated. Experiments using agonistic and neutralizing anti-Fas antibodies indicated that Fas-mediated signaling was not operative in vehicle or myeloid cells exposed to O6BG/TMZ. These data indicate that 6BG/TMZ-mediated apoptosis is initiated predominantly via the intrinsic-mitochondrial apoptotic pathway- and that while Fas is expressed on human myeloid cells, it does not contribute to cell death in myeloid cells. This model system provides a feasible approach to probe further how hematopoietic cells respond to therapy and to search for new strategies that will maintain genome stability in primary hematopoietic cells during anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4671. doi:1538-7445.AM2012-4671