Diminished Oxidative Stress Responses in Bone Marrow Stromal Cell Lines Derived From Fanconi Anemia (Fanc-D2−/−) Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4398-4398
Author(s):  
Hebist Berhane ◽  
Michael W. Epperly ◽  
Shaonan Cao ◽  
Xichen Zhang ◽  
Donna Shields ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Dna Repair ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Stromal Cell ◽  
Whole Body ◽  
Hematopoietic Progenitor Cell ◽  
Marrow Cultures

Abstract Abstract 4398 Fanconi Anemia (FA) is an inherited cancer susceptibility syndrome associated with a defect in one or more of the 15 FA pathway gene products leading to bone marrow failure, increased cellular sensitivity to DNA cross-linking agents and increased risk of myeloid leukemia. Underlying DNA repair vulnerabilities render FancD2−/−, a homozygous deletion recombinant negative mice, sensitive to whole body irradiation. We quantitated hematopoiesis in long term bone marrow cultures, and the radiosensitivity and oxidative responses of derived marrow stromal and IL-3 dependent hematopoietic progenitor cell lines derived from FancD2−/− mice on a C57BL/6 background (generously provided by Drs. A. D'Andrea and K. Parmar, Dana-Farber cancer Inst., Boston, MA). Long-term bone marrow culture stromal cell lines derived from FancD2−/− were compared to FancD2+/− or FancD2+/+. FancD2−/− long term marrow cultures showed decreased cobblestone islands, representative of hematopoietic stem cell generating foci, and production of non-adherent cells compared to wild type FancD2+/+ or heterozygous FancD2 +/− cultures (p = 0.0148) and produced fewer colony forming cells on day 7 and 14 compared to FancD2 +/− or FancD2 +/+ cell lines (p < 0.0001). FancD2−/− stromal cell lines showed decreased DNA repair by comet assay after irradiation to 5Gy (P<0.0001). Following irradiation to 5 or 10 Gy, FancD2−/− cells had an increased cell doubling time (44.1 + 0.7 and 54.2 + 7.8) compared to FancD2 +/+ cells (35.5 + 1.5hrs and 33.6 + 1.3,hrs p = 0.006 and 0.051, respectively). FancD2−/− cells had decreased baseline antioxidant levels compared to the FancD2 +/+ cells (0.55 + 0.11 and 0.91 + 0.17 trolox units, respectively). FancD2−/− cells at 24 hr following 10 Gy irradiation had 0.39 + 0.03 trolox units compared to 0.76 + 0.07 trolox units in FancD2 +/+ cells (p = 0.006). In clonogenic irradiation survival curve assays, FancD2−/− marrow stromal cells were radiosensitive (Do = 1.43 + 0.06 Gy, n = 4.98 + 0.65) compared to FancD2 +/+ (Do 1.70 + 0.09 Gy and n = 8.33 + 0.72, p = 0.0395 and 0.0040, respectively) or FancD2 +/− cells (Do = 1.67 + 0.08 and n = 3.63 + 0.44, p = 0.0348 and 0.1365, respectively). Furthermore, FancD2 +/− cells were more radiosensitive compared to FancD2 +/+ cells ((n = 3.63 + 0.44 or 8.33 + 0.72), p = 0.0003). Thus, the FancD2 gene controls several parameters of sensitivity to oxidative stress and ionizing radiation as demonstrated in mouse long-term marrow cultures and derived cell lines. Disclosures: No relevant conflicts of interest to declare.

DNA Cross-Linking Agent Sensitivity of Fanconi Anemia (FA) Cells Is Preserved in Double Knockout (DKO) SMAD3-/- Fancd2-/- Mouse Cell Lines

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4799-4799
Author(s):  
Zean Chen ◽  
Darcy Franicola ◽  
Donna Shields ◽  
Michael W. Epperly ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mitomycin C ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Marrow Stromal Cells ◽  
Cross Linking ◽  
Increased Sensitivity ◽  
Marrow Cultures

Abstract Increased sensitivity to inhibition of hematopoiesis by TGF-β has been hypothesized to be a mechanism of hematopoietic failure in Fanconi Anemia (FA). To determine whether abrogation of TGF-β signaling rescued biologic parameters of hematopoiesis, we derived a novel DKO mouse strain (SMAD3-/- (129/Sv) Fancd2-/- (C57BL/6)) by breeding double heterozygote mice. The DKO mice were small at birth, but achieved normalized growth and development by six - eight weeks. Hematopoiesis in long-term bone marrow cultures derived from DKO mice, ceased generating day 14 CFU-GEMM hematopoietic progenitors by 18 weeks, similar to that observed with Fancd2-/- marrow cultures. In contrast, cultures from SMAD3-/- (129/Sv) demonstrated continuous hematopoiesis for over 30 weeks confirming results with marrow from SMAD3-/- (C57BL/6J) mice (Epperly, et al., Experimental Hematology, 33:353-362, 2005), and significantly longer than that in control 129/Sv X C57BL/6J F1 mouse long-term marrow cultures. Bone marrow stromal cell lines derived from long-term marrow cultures were tested for the diagnostic criterion of FA, increased sensitivity to DNA cross-linking agent, Mitomycin-C. Cells were incubated in Mitomycin-C at concentrations ranging from 0-20 ng/ml for four days and then plated in 4 well tissue culture plates, incubated for 7 days at 37o C in a CO2 incubator, stained with crystal violet, and colonies of greater than 50 cells counted. Similar to Fancd2-/- marrow stromal cells, DKO marrow stromal cells showed increased sensitivity to Mito-C, while marrow stromal cell lines from wild type or SMAD3-/- mouse marrow cultures were relatively resistant (p = 0.0086 comparing Fancd2-/- to control 129/Sv X C57BL/6J F1 cells, p = 0.0156 comparing DKO with control 129/Sv X C57BL/6J F1 cells, and p = 0.4676 comparing Smad3-/- cells to control 129/Sv X C57BL/6J F1 cells). Fresh bone marrow from DKO mice, similar to that from SMAD3-/- mice, demonstrated resistance to inhibition of hematopoietic cell colonies in vitro by increasing concentrations of TGF-β. Therefore, abrogation of TGF-β signaling in DKO cells did not alter the sensitivity of Fancd2-/- marrow stromal cells to Mitomycin-C. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

Biology of Marrow Stromal Cell Lines Derived from Long-Term Bone Marrow Cultures of Trp53-Deficient Mice

1999 ◽  
Vol 152 (1) ◽  
pp. 29 ◽  
Author(s):  
Michael W. Epperly ◽  
Jenifer A. Bray ◽  
Timothy M. Carlos ◽  
Edward Prochownik ◽  
Joel S. Greenberger
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Marrow Stromal Cell ◽  
Bone Marrow Cultures ◽  
Deficient Mice ◽  
Marrow Cultures

scholarly journals Transformed Phenotype of Bone Marrow Stromal Cell Lines Derived from K14E7 Fancd2-/- mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4795-4795
Author(s):  
Aranee Sivanathan ◽  
Xichen Zhang ◽  
Darcy Franicola ◽  
Shaonan Cao ◽  
Donna Shields ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Wild Type ◽  
Marrow Stromal Cell ◽  
Cytokeratin 14 ◽  
E7 Oncogene ◽  
Marrow Cultures

Abstract To determine whether cytokeratin 14 promoter linked expression of the Human Papilloma Virus (HPV) oncogene detectably influenced biologic parameters of cell phenotypes other than squamous epithelium, continuous bone marrow cultures were derived from K14E7 Fancd2-/- mice (Park, et al., Cancer Research, 70(23): 9959-9968, 2010). Long-term bone marrow cultures derived from K14E7 Fancd2-/-, control Fancd2-/- (129/Sv), K14E7 (FVB/N), and wild type 129/Sv X FVB/N F1 mice were evaluated for longevity of hematopoiesis in long-term cultures and stromal cell lines were derived from each. Similar to Fancd2-/- mouse, long-term marrow cultures, K14E7 Fancd2-/- marrow cultures demonstrated decreased longevity of hematopoiesis with cessation of production of multi-lineage colony forming progenitor cells after 14 weeks. In contrast, wild type F1 and K14E7 long-term marrow cultures continued to produce hematopoietic cells for a significantly longer duration 25 weeks (p=0.0257). Bone marrow stromal and IL-3 dependent hematopoietic cell lines were derived from each genotype marrow culture. K14E7 Fancd2-/- hematopoietic cells showed reversal of the radiation resistance of Fancd2-/- IL-3 dependent cell lines (D0 of 1.34 ± 0.197Gy, ñ 4.0 ± 0.9 compared to D02.213 ± 0.124 Gy (p = 0.0284), ñ 3.3 ± 0.8. Thus, one phenotypic difference associated with K14E7 oncogene expression was reversal of radioresistance of Fancd2-/- hematopoietic cells. In contrast, bone marrow stromal cell lines from K14E7 Fancd2-/- remained radiosensitive similar to those from Fancd2-/- mice. K14E7 and wild type F1 marrow stromal cell lines showed intermediate radioresistance (p= 0.1759). To determine whether the E6/E7 oncogene had a biological effect in tissues other than squamous epithelium, tissue analysis for cytokeratins 13, 14, 6, and 10 was carried out. Cytokeratin 14 was detected only in squamous cells of the esophagus and oral cavity, not in bone marrow. E6/E7 oncogene was detected only in squamous cell lines expressing cytokeratin 14. However, bone marrow stromal cell lines from K14E7 Fancd2-/- marrow cultures demonstrated a unique phenomenon of cellular density, piling up and formation of tumors in vitro. Each of 22 single cell derived clonal sub-lines of K14E7 Fancd2-/- stromal cell lines demonstrated the same transformed phenotype. These data provide support for indirect effects of the E7 oncogene linked to the K14 promoter in Fancd2-/- hematopoietic and mesenchymal stem cell tissues. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

Long-Term Bone Marrow Cultures: Recent Studies with Clonal Hematopoietic and Stromal Cell Lines

1989 ◽  
Vol 8 (1) ◽  
pp. 1-20 ◽  
Author(s):  
Masatsugu Ohta ◽  
Pervin Anklesaria ◽  
T.J. FitzGerald ◽  
Kenneth Kase ◽  
Jean Leif ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

scholarly journals Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2269-2277 ◽  
Author(s):  
HM Lokhorst ◽  
T Lamme ◽  
M de Smet ◽  
S Klein ◽  
RA de Weger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Adherent Cells ◽  
Myeloma Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

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

2014 ◽  
Vol 181 (1) ◽  
pp. 76 ◽  
Author(s):  
Hebist Berhane ◽  
Michael W. Epperly ◽  
Julie Goff ◽  
Ronny Kalash ◽  
Shaonan Cao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor
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