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.

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 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

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 Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

scholarly journals Bone marrow stromal cells modulate both kappa light chain and Ly1 antigen expression on Ly1+ pre-B cell lines in vitro

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 383-392 ◽  
Author(s):  
MG Kruger ◽  
RL Riley ◽  
EA Riley ◽  
JM Elia
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Light Chains ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Murine Ly1+ pre-B cell lines, including 70Z/3 and three pre-B cell lines derived from long-term bone marrow cultures, exhibited selective adherence to bone marrow stromal cells. In contrast, splenic B cells, the A20 B-cell lymphoma, and four Ly1- B cell lines derived from long- term bone marrow cultures failed to adhere substiantially to bone marrow cultures failed to adhere substiantially to bone marrow stroma. Ly1+ pre-B cell lines were induced to express kappa light chains by exposure to either lipopolysaccharide (LPS), recombinant interleukin-1 (IL-1), or stromal cells. However, induction of kappa light chains failed to prevent pre-B cell adherence to stromal cells. Supernatants derived from primary bone marrow stromal cells decreased Ly1 expression on the Ly1+ pre-B cell lines. These experiments suggest that (1) expression of immunoglobulin light chains by developing Ly1+ pre-B cells is mediated by bone marrow stromal cells; (2) loss of specific adherence to stroma is progressive and occurs post-light chain induction; and (3) soluble products of stromal cells may downregulate expression of surface Ly1 on otherwise Ly1+ pre-B cells. The importance of these observations to the development of both the Ly1- and Ly1+ B cell lineages in the mouse is discussed.

scholarly journals Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089 ◽  
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

Abstract The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

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

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.

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