Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2–/–) Mice

Abstract

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.

Induction of TGF-β by Irradiation or Chemotherapy in Fanconi Anemia (FA) Mouse Bone Marrow Ιs Modulated by Small Molecule Radiation Mitigators JP4-039 and MMS350

“…Fresh bone marrow was explanted from the mouse femur and tibia at serial time points after administration of chemotherapy drugs and plated in 6-well culture plates at 5×10 4 cells/per well for hematopoietic colony assays according to published methods (34). The medium was supplemented with hematopoietic growth factors as described (29) to stimulate multilineage hematopoietic stem cell growth.…”

“…The medium was supplemented with hematopoietic growth factors as described (29) to stimulate multilineage hematopoietic stem cell growth. Colonies of greater than 50 cells were scored at days 7 and 14 (34).…”

“…Bone marrow stromal cell lines (34) and IL-3 dependent hematopoietic progenitor cell lines (34,36) were established from control C57BL/6NTac, FANCD2 -/-and FANCD2 +/-(B6) long-term bone marrow cultures according to published methods (34). Bone marrow stromal cell lines were established from TGF-β -/-newborn mice on day one after birth according to published methods (34), or from the adherent layer of four week old long-term bone marrow cultures from TGF-β +/-and TGF-β +/+ control (C57BL/6NTac) mice according to published methods (31). Clonal sublines used were designated TGF-β +/+ 2A6, TGF-β +/-2C9, and TGF-β -/-2C7.…”

“…IL-3 dependent cell lines derived from long term bone marrow cultures that were established from FANCD2 -/-and FANCD2 +/+ mice have been previously reported (34). Each cell line was passaged in suspension culture in IL-3 supplemented medium according to published methods (34,36).…”

“…FANCD2 -/-mouse marrow hematopoietic progenitors in vitro and stromal cell lines derived from the hematopoietic microenvironment (34) were used for TGF-β induction by irradiation and chemotherapy drugs. We also tested the effect of JP4-039 and MMS350 on TGF-β induction in plasma of TBI irradiated or drug-treated FANCD2 -/-(C57BL/6 background) mice.…”