a Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213;
Radiat Res. 2014 Jan;181(1):76-89. doi: 10.1667/RR13405.1. Epub 2014 Jan 7.
FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2(-/-) mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2(-/-) mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2(-/-) mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2(+/+) stromal cells (Fancd2(-/-) D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2(+/+) D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2(-/-) IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2(+/+) (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2(-/-) marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2(-/-) stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2(+/+) cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2(-/-) IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2(-/-) stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.
FancD2 在人类范可尼贫血 DNA 损伤反应 (DDR) 途径中发挥核心作用。 Fancd2(-/-) 小鼠表现出许多人类范可尼贫血的特征，包括细胞 DNA 修复缺陷。 Fancd2(-/-) 小鼠中的 DNA 修复缺陷是否会导致所有细胞谱系的放射影像学改变尚不清楚。我们测量了长期骨髓培养物中的造血应激和集落形成存活曲线中的放射敏感性，以及与骨髓基质细胞系相比，造血祖细胞中的彗星尾强度、总抗氧化储存量和辐射诱导的基因表达。我们进一步评估了线粒体靶向抗氧化剂 GS-氮氧化物 JP4-039 的放射保护作用。与 Fancd2(+/+) 基质细胞相比，Fancd2(-/-) 小鼠长期骨髓培养物中的造血寿命缩短，骨髓基质细胞系对辐射敏感 (Fancd2(-/-) D0 = 1.4 ± 0.1 Gy，ñ = 5.0 ± 0.6 与 Fancd2(+/+) D0 = 1.6 ± 0.1 Gy，ñ = 6.7 ± 1.6)，P = 0.0124 用于 D0 和 P = 0.0023 用于ñ)。相比之下，Fancd2(-/-) IL-3 依赖性造血祖细胞对辐射具有抗性 (D0 = 1.71 ± 0.04 Gy 和 ñ = 5.07 ± 0.52)，而 Fancd2(+/+) (D0 = 1.39 ± 0.09 Gy 和 ñ = 2.31 ± 0.85，P = 0.001 用于 D0)。从新离体的 Fancd2(-/-) 骨髓中分离出的 CFU-GM 也具有抗辐射性。与放射敏感性一致，与 Fancd2(+/+) 细胞相比，辐照的 Fancd2(-/-) 基质细胞的彗星尾强度测定法具有更高的 DNA 损伤 (P < 0.0001)，DNA 损伤恢复较慢，基础总抗氧化能力较低，辐射诱导的抗氧化剂耗竭增强，以及 CDKN1A-p21 基因转录本和蛋白质增加。与放射抗性一致，Fancd2(-/-) IL-3 依赖性造血细胞具有更高的基础和辐照后总抗氧化能力。虽然 Fancd2(-/-) 基质细胞中未检测到辐射诱导的细胞周期停滞的改变，但造血祖细胞显示出减少的 G2/M 细胞周期停滞。缺乏小鼠 Fancd2 基因产物使骨髓基质细胞而非造血祖细胞对辐射敏感。
