Abstract
Aim: To determine if the small-molecule radioprotector GS-nitroxide, JP4-039, improved hematopoiesis in long-term bone marrow cultures (LTBMCs), explanted marrow from in vivo drug-treated C57BL/6NTac mice was maintained in JP4-039 for 25 weeks. Hematopoietic cell production and radiobiology of derived stromal cell lines was measured. Materials and Methods: Groups of LTBMCs were established from mouse groups. Stromal cell lines were established from the adherent layer of JP4-039-treated and untreated control groups. Results: LTBMCs maintained in JP4-039 exhibited increased production of total non-adherent and 7-day and 14-day hematopoietic colony-forming cells. Stromal cell lines derived from JP4-039-treated cultures were radioresistant in vitro, demonstrated a distinct squamous/epithelial morphology and overexpressed Nrf2, Ctgf, Lox, Tlr1, collagen 1a, Brd3, and Brd4. Conclusion: Chronic treatment of bone marrow cultures and derived stromal cell lines with JP4-039 was non-toxic, and conferred resistance to oxidative stress.
A major concern for the use of potential radioprotective and radiation-mitigating drugs is their safety. Mouse long-term bone marrow cultures (LTBMCs) are a useful system to assess toxicity of a variety of agents including oxidative stress on hematopoiesis in vitro (1-6). Prior studies have demonstrated that the administration of GS-nitroxide JP4-039, a mitochondrial-targeted tempol, by intravenous, intraperitoneal, or swallowed route is associated with no acute toxicity (7-8, 10-14). In recent studies, intra-oral administration of JP4-039 in a localized emulsion was demonstrated to successfully protect the esophagus from irradiation (13) with no detectable systemic toxicity.
We have represented the potential value of the GS-nitroxide drug, JP4-039, as a radiation protector and mitigator (7-8, 10-13). One concern for use of JP4-039 as a radioprotective or radiation-mitigating small molecule is late toxicity. In the present studies, we tested the effect of continuous administration of JP4-039 for 25 weeks on oxidative stress from LTBMCs. Hematopoietic and mesenchymal stem cell (bone marrow stromal cell) lines derived from the adherent layer of bone marrow cultures were tested for markers of toxicity (1-2, 9).
Materials and Methods
Mice. C57BL/6NTac mice (Taconic Farms, Hudson, NY, USA) were housed five per cage according to University of Pittsburgh Institutional Animal Care And Use Committee (IACUC) regulations and fed standard Purina laboratory chow. A subgroup of mice received JP4-039 i.v. at 20 mg/kg weekly for two weeks before marrow explant. All protocols were approved by the University of Pittsburgh IACUC. Veterinary care was provided by the Division of Laboratory Animal Research of the University of Pittsburgh.
LTBMC. LTBMCs were established from the femur and tibia marrow of C57BL/6NTac mice as described elsewhere (1, 2). The contents of a femur and tibia (n=6/genotype) were flushed into McCoy's 5A medium (Gibco, Gaithersburg, MD, USA) supplemented with 25% horse serum (Cambrex, Rockland, ME, USA), and 10−5 M hydrocortisone sodium hemisuccinate. Cultures were incubated at 33°C in 7% CO2. After four weeks, the horse serum was replaced with 25% fetal bovine serum (FBS) (Gibco) (1, 2). The cultures were examined weekly for hematopoietic cell production and cobblestone island formation. Cobblestone islands of 50 cells or more were scored weekly in each flask (1, 2). A two-sided two-sample t-test was used to compare the number of cobblestone islands between cultures each week. p-Values less than 0.05 were regarded as significant. JP4-039 was added weekly to some cultures at 10 μM as described elsewhere (11). As a vehicle control 1 μl/ml DMSO was added weekly to the cultures.
Hematopoietic cell colony-forming assays. A total of 1×105 non-adherent cells were removed from the LTBMC flask and 5×104 cells/dish were plated in triplicate in semi-solid medium consisting of methylcellulose in Iscove's Modified Dulbecco's medium (IMDM) supplemented with FBS, 10% bovine serum albumin (BSA), L-glutamine, 3 U/ml erythropoietin and 2-meraptoetanol (Stem Cell Technology, Vancouver, CA). Colony-forming unit granulocyte-macrophage (CFU-GM) of 50 cells or greater were counted on days 7 and 14 after plating. A two-sided, two-sample t-test was used as described above.
Bone marrow stromal cell lines and clonal cell sublines. Adherent cell layers from one 4-week-old LTBMC were trypsinized and expanded by passage into Dulbecco's modified Eagle's medium with 10% FBS to establish bone marrow stromal cell lines according to published methods (9). Cells were passaged for 10 weeks to establish cell lines. Cultures were incubated at 37°C in 5% CO2.
Clonogenic irradiation survival curves for mouse bone marrow stromal cell lines. Bone marrow stromal cells were irradiated in suspension to doses between 0 and 8 Gy at 70 cGy/min using a Shepherd Mark 1 137Cs γ-ray source (J.L. Shepherd, San Fernando, CA, USA). Cells were plated in quadruplicate in Linbro plates (Fisher Scientific, Pittsburgh, PA, USA) and incubated at 37°C and 5% CO2 for 9-11 days, stained with crystal violet, and colonies of ≥50 cells were counted using a GelCount colony counter (Oxford Optronix, Oxford, UK). Data were analyzed with single-hit multitarget model according to published methods (6, 10-11).
Reverse transcription polymerase chain reaction (RT-PCR) analysis. Unirradiated cells from each stromal cell line were harvested. Real time polymerase chain reaction (RT-PCR) was used to analyze radiation-inducible transcripts for transcription factors nuclear factor kappa-light-chain-enchancer of activated B cells (Nfkb) and nuclear factor (erythroid-derived 2)-like2 (Nrf2), and cytokines including transforming growth factor-beta one (Tgfb1), for the oxidative stress response enzyme manganese superoxide dismutase MnSOD (Sod2), and for irradiation response-related transcripts p21 and p53 using published methods (10). The results are presented as fold increase in gene expression above baseline level, which was adjusted to that for non-irradiated C57BL/6NTac wild-type mouse bone marrow stromal or hematopoietic progenitor cells. Total RNA was extracted from cell pellets using Trizol reagent (Invitrogen). A total of 2 μg of RNA was used to synthesize cDNA in a 20 μl reaction system, according to the instructions of the high-Capacity cDNA Reverse Transcription Kit (cat# 4368814; A&BApplied Biosystems, Foster City, CA, USA) with the reaction conditions of 40 cycles of 95°C (denaturation) for 15 s and 60°C (annealing and elongation) for 1 min using the Eppendorf Realplex2 Mastercycler (Westbury, NY, USA). For RT-PCR, reaction conditions were as follows: Ninety-six-well plates were prepared with 10 μl of Taqman Gene Expression Master mix (A&B Applied Biosystems, Foster City, CA, USA), 5 μl of RNase-free water, 1 μl of the corresponding Taqman Gene Expression probe, and 4 μl of cDNA (totaling 2 μg of cDNA) using the Eppendorf epMotion 5070 automated pipetting system (Eppendorf, Westbury, NY, USA). PCR amplification of the Gapdh gene was used as the house-keeping gene (Gen-Bank: NM_008084). Genes analyzed are shown in Table I.
Statistical methods. The in vitro radiation survival curves were analyzed with the single-hit multitarget model, and were compared using D0 (final slope representing multiple-event killing) and ñ (extrapolation number measuring width of the shoulder on the radiation survival curve) (8). Results for D0 and ñ are presented as the mean±standard error (SEM) from multiple measurements and compared with the two-sided two-sample t-test.
For LTBMC data, weekly cobblestone island numbers, non-adherent cell numbers (×105); percentage confluence of adherent cells, day 7 colony-forming cells at each weekly harvest, and day 14 colony-forming cell count at each weekly harvest were counted as described elsewhere (6). At each week, the cobblestone number collected from two flasks, were calculated by averaging the two numbers. These averages were summarized as the mean±standard deviation, and p-values were calculated with the two-sided two-sample t-test on these averages. Similar calculations and tests were performed for the estimation of non-adherent cell numbers and the percentage of confluence of adherent cells The colony count data were also compared between JP4-039-treated and untreated control at each week with the two-sided two-sample t-test. As this was an exploratory study, p-values were not adjusted for multiple comparisons.
Results
Longevity of hematopoiesis in LTBMCs. LTBMCs were established from control or JP4-039 treated C57BL/6NTac mice. As shown in Figure 1A, the percentage confluence of the adherent stromal layer in LTBMCs was unchanged by the presence of JP4-039. The cobblestone islands per flask (Figure 1B), which are a reflection of the number of hematopoietic cell islands consisting of 50 or more flattened cells per island, significantly increased in JP4-039-treated LTBMCs, compared to the control.
As shown in Figure 1C, the cumulative production of cobblestone islands was significantly increased in JP4-309-treated l LTBMCs.
We next measured non-adherent cells produced weekly per flask, and results are shown in Figure 1D. There was an increase in production of non-adherent cells between weeks 9 and 15 in JP4-039-treated LTBMCs. This weekly cell production led to an increased cumulative non-adherent cell production in the JP4-039-treated group (Figure 1E).
A prominent feature of continuous hematopoiesis in LTBMCs is the production of colonies of hematopoietic cells in secondary culture. As shown in Figure 1F, JP4-039-treated cells, when removed and plated at 5×104 cells per dish in semi-solid medium, formed 50-cell colonies at day 7 which significantly increased between weeks 2 and 12 (Figure 1F). The cumulative day 7 colony-producing cell number was also significantly increased in JP4-039-treated cultures (Figure 1G). The more primitive hematopoietic colony-forming cells, which grow more slowly and produce more than 50 cell colonies by day 14, are a reflection of the stability of LTBMCs and the in vitro longevity of hematopoietic progenitors capable of prolonged survival in the adherent layer. These cells are more slowly released into the nonadherent layer and are measured by the day 14 colony assay. As shown in Figure 1H, weekly production of day 14 colony-forming progenitor cells was significantly increased in JP4-039-treated LTBMCs between weeks 2 and 12. Cumulative production of these more primitive hematopoietic progenitors was also significantly increased in the presence of JP4-039 (Figure 1I).
Genes analyzed comparing C57BL/6-JP4-039 and C57BL/6 bone marrow stromal cell lines.
Increased radioresistance of bone marrow stromal cells derived from JP4-039-treated LTBMCs. The establishment of permanent clonal bone marrow stromal cell lines from JP4-039-treated and control bone marrow cultures was carried out according to published methods. Stromal cell lines were expanded in culture and clonal sublines were derived. The radiation sensitivity in a clonogenic survival curve was carried out according to published methods (9). Colonies formed by single cells plated at varying plating densities were scored after radiation to doses ranging between 0 and 8 Gy. The colonies of over 50 cells per adherent colony were scored on day 7. As shown in Figure 2, stromal cells derived from a JP4-039-treated LTBMCs were intrinsically radioresistant (C57BL/6-JP4-039). The statistical analysis of these cells showing greater radioresistance is shown in Table II. Stromal cell lines from control bone marrow cultures exhibited intrinsic relative radiosensitivity; however, when grown in the presence of JP4-039 100 μM added either prior to irradiation or post-irradiation, the cells were also relatively radioresistant (Figure 2, Table II).
Increased radioresistance of bone marrow stromal cell line derived from long term bone marrow cultures (LTBMCs) from C57BL/6-JP4-039 treated mice.
The morphology of bone marrow stromal cells from JP4-039-treated LTBMCs is distinct from that of control marrow cultures. The photographs of stromal cell lines derived from JP4-039-treated LTBMCs are shown in Figure 3. Stromal cells derived from control LTBMCs exhibited the classic morphology of marrow adherent cells in long-term culture, with stellate, endothelial, and mesenchymal cell morphology. These cells migrated across the culture dish and colonies were diffuse, with separation between them (Figure 3A). In contrast, the stromal cell line derived from JP4-039-treated LTBMCs formed sheets of adherent cells with flattened morphology and more uniform epithelial morphology. These are more classic of embryonic fibroblast cell lines, and suggest the production of adhesion molecules, establishment of tight junctions, and lack of either response/production of scatter factor (hepatocyte growth factor), which has been shown to produce the diffuse morphology of colonies derived from LTBMC adherent cells (Figure 3B).
The morphology of a single cell-derived colony in the radiation survival curve assay using cells from control long-term bone marrow culture stromal cell lines is shown in Figure 3C. These cells exhibited the migratory pattern of single cells in the colony assay and the distinct shapes of stellate morphology. In contrast, a hematoxylin and eosin-stained colony from a JP4-039-treated LTBMC-derived stromal cell line is shown in Figure 3D and demonstrates the presence of tight junctions and uniform flattened morphology. These data suggest that prolonged treatment with JP4-039 altered bone marrow stromal cell biology and/or selected a population of cells more typical of epithelial cells.
Hematopoiesis in long-term marrow cultures: A: percentage confluence; B: cobblestone islands; C: cumulative cobblestone islands; D: nonadherent cell production per flask weekly; E: cumulative nonirradiated cell production; F: weekly production of day 7 colony-forming cells; G: cumulative production of day 7 colony-forming cells; H: weekly production of day 14 colony-forming cells; and I: cumulative production of day 14 colony-forming cells. Significant differences between C57BL/6-JP4 and C57BL/6 LTBMCs are indicated by *(p<0.05).
Radiation survival curve of stromal cells chronically treated with JP4-039. Bone marrow stromal cell lines were established from C57BL/6NTac mice-injected with JP4-039 weekly for two weeks before isolation of marrow and maintained in JP4-039 (10 μM) constantly for 25 weeks in long-term bone marrow cultures (LTBMC) then for eight additional weeks or from control C57BL/6NTac mice never exposed to JP4-039. In vitro irradiation survival curves were performed as described in materials and methods. JP4-039 (Pre) are C57BL/6 stromal cell lines exposed to JP4-039 (10 μM) 1 h before irradiation, and JP4-039 (Post) are C57BL/6 stromal cells exposed to JP4-039 (10 μM) immediately after irradiation, and during culture for colony assay. There was radioprotection or mitigation (post) in all JP4-039-treated cell lines. Statistical analysis of irradiation survival curves are shown Table II.
Gene transcript levels in JP4-039-treated LTBMC-derived stromal cells are distinct from those derived from control bone marrow cultures. We tested the expression of RNA transcript levels for a variety of genes found previously to be involved in bone marrow stromal cell responses to oxidative stress. These include categories of adhesion molecules including Tlr1-7, bromodomain epigenetic reader proteins Brd1-4, and Col1a, as well as a variety of growth factors including Tnfα, Tgfβ, Il6, and gene promoter binding proteins including Nf-κb and Nrf2. As shown in Figure 4, JP4-039-treated LTBMC-derived stromal cells exhibited a significant increase in Nrf2, and Ctgf expression levels (Table II). Transcript levels of Lox, Tlr1, Col1a, as well as Brd3, and Brd4 were many fold higher compared to those of control cell lines. The marked increase in expression of Col1a may be correlated to the epithelial morphology of this cell line. Of interest, expression of Nf-kb, encoding an irradiation-induced promoter-binding protein associated with response to oxidative stress, was decreased. There were 15 gene transcripts that were significantly decreased in JP4-039-treated stromal cell line, (Table II, Figure 4).
Inverted microscopic and histological appearance of stromal cell line colonies in vitro. A: Control C57BL/6 (×100); B: C57BL/6-JP4-039-treated culture stromal cells (×100); C: hematoxylin and eosin-stained C57BL/6 colony used for the data shown in Figure 2 (original magnification, ×50); D) hematoxylin and eosin stained C57BL/6-JP4-039 colony (original magnification, ×50).
RNA transcripts in JP4-039-treated mouse marrow stromal cell line derived from long-term bone marrow cultures (LTBMCs). Results are standardized to those for control marrow stromal cell line. C57BL/6-JP4-039 bone marrow stromal cells were derived from LTBMCs from mice injected with 10 μM JP4-039 once a week for two weeks before isolation of bone marrow and constantly for four weeks in LTBMC and eight weeks as a cell line. C57BL/6 bone marrow stromal cell line was derived from a LTBMC made from C57BL/6 marrow of mice never exposed to JP4-039 in vitro or in vivo. RNA was extracted and gene expression was determined using real time-polymerase chain reaction (RT-PCR). Data are expressed for the C57BL/6-JP4-039 relative to control C57BL/6 LTBMC cell lines. Significant differences in relative gene expression comparing C57BL/6-JP4-039 to C57BL/6 are shown (*p<0.05).
Discussion
The mouse LTBMC system has been demonstrated to be a useful model for testing the effects of cytotoxic, inflammatory, and infectious agents on the interaction between hematopoietic stem cells and bone marrow stromal cells (1-4, 8-9).
While other organ explant systems have been reported, the duration of hematopoiesis in continuous bone marrow cultures (reaching over one year in some mouse strains) (2) has provided a unique opportunity for dissecting the molecular mechanism of interaction between self-renewing stem cell populations and cells of their microenvironment. In the present study, we tested the hypothesis that the oxidative stress of long-term culture was one of the components limiting longevity of hematopoiesis in continuous bone marrow cultures, and that continuous addition of an antioxidant to the system would improve the longevity of hematopoiesis.
GS-nitroxides represent a recently reported class of small-molecule electron and reactive oxygen species (ROS) scavengers benefiting from mitochondrial targeting and enrichment (5, 7, 11-13). Previous studies have demonstrated that the localization of antioxidants to the mitochondria stabilizes mitochondrial biochemistry, reducing mitochondrial membrane permeability, and the leakage of cytochrome c into the cellular cytoplasm, which initiates the caspase system and leads to apoptosis (8). In particular, JP4-039 has been demonstrated to increase radioresistance of isolated hematopoietic and stromal cells in culture (10), and limit bone marrow cytotoxicity from total body irradiation in vivo (7).
In the present studies, we added JP4-039 to continuous bone marrow cultures derived from C57BL/6NTac mice weekly for 25 weeks in vitro. We hypothesized that continuous supplementation of culture media with antioxidant would reduce apoptosis, and stabilize interactions between hematopoietic cells and cells in the hematopoietic microenvironment. The study demonstrated an improved longevity of hematopoiesis in JP4-039-supplemented LTBMCs. Improved hematopoiesis was reflected in both the stability of the adherent layer, the numbers of cobblestone islands, indicative of adherent multi-lineage hematopoietic stem cell progenitors, as well as the cumulative production of non-adherent cells, and cells forming 7 day and 14 day multi-lineage hematopoietic colonies in secondary semi-solid medium culture. These data establish that JP4-039 is nontoxic to hematopoiesis in long-term culture in vitro and stimulates longevity of hematopoiesis. The multi-lineage day 14 CFU-GEMM colonies derived from JP4-039-treated long-term bone marrow cultures maintained multi-lineage differentiation capacity in secondary culture, suggesting that the effect of JP4-039 on stromal cells in long-term culture did not limit the capacity of stromal cells to support multi-lineage hematopoietic stem cells.
The morphology of colonies in day 7 and day 14 colony assays was typical of those in control marrow cultures. Thus, our results provide strong evidence that the increased longevity of hematopoiesis with the production of hematopoietic cells correlates with in vivo mitigation of ionizing irradiation injury from total body irradiation (7). Further studies quantitating the continuous presence of true stem cells, forming CFUs in vitro, or having enhanced long-term repopulating capacity in competitive repopulation assays, should provide additional insight into this hypothesis.
Cell lines derived from the adherent layer of long-term cultures that were maintained in JP4-039 and continuously grown in the presence of JP4-039 demonstrated an intrinsic radiation resistance in a clonogenic survival curve. Bone marrow stromal cell lines derived from control long-term bone marrow cultures were relatively radiosensitive; however, addition of JP4-039 to control bone marrow stromal cell lines induced radioresistance whether it was added before or after irradiation, demonstrating both radioprotection and radiation mitigation. The intrinsic radioresistance of cells continuously maintained in JP4-039 prior to radiation suggests that the drug alters one or more intrinsic properties of the cellular response to irradiation.
One observation from the present studies was the change in morphology of the bone marrow stromal cells derived from cultures grown in JP4-039, both as cell lines, and as colonies, as shown in clonogenic radiation survival curves. JP4-039-treated bone marrow stromal cells demonstrated novel adherence of cells to each other, growing in sheets typical of squamous epithelium in vitro, while control culture-derived bone marrow stromal cell lines demonstrated a migratory capacity in vitro that is typical of mesenchymal cells. Further studies are required to determine whether the squamous morphology of JP4-039 culture-derived stromal cells is reflective of differentiation, or due to a more primitive mesenchymal stem cell biology. The morphology of bone marrow stromal cell lines derived from LTBMCs that have been maintained in JP4-039 was different from those kept in control cultures. Cultures maintained in JP4-039 exhibited adherent cell bridges and sheets of cytokeratin-positive-appearing keratinocytes more typical of embryonic stem cells than those of bone marrow stromal cells. Photomicrographs of control LTBMCs were typical of those previously published (2) and demonstrate an admixture of endothelial appearing cells, fibroblasts, and macrophages. In contrast, cell lines derived from JP4-039-treated cultures demonstrated a uniform morphology with cellular bridges and flattened areas more typical of organ cultures. Whether adhesion molecule production by stromal cells in culture is stimulated by JP4-039 will require further investigation. Irradiation survival curves of bone marrow stromal cells derived from JP4-039-treated cultures demonstrated intrinsic radioresistance. Furthermore, treatment of control bone marrow stromal cells in vitro with JP4-039, whether prior to or after irradiation, increased radioresistance. These results confirm and extend those from previous publications indicating that JP4-039 added to cells acutely before or after irradiation is a radioprotector or radiation mitigator, respectively (8). Further studies may include: analysis of expression of cell surface adhesion molecules, cytokines and their receptors, and ultrastructural analysis of the bridges holding cells together.
The present studies provide further support for the use of JP4-039 as a clinically-relevant radiation protector and radiation mitigator in larger animal species, including humans, due to lack of toxicity and the potential capacity to support the integrity of both stem cell populations and cells of their microenvironment.
Notably, adding the GS-nitroxide to murine LTBMCs significantly increases the production of cobblestone islands, representative of hematopoietic adherent cell dividing units, as well as the cumulative production of hematopoietic cells. The cumulative production of cells forming day 7 CFUs in culture and those cells forming day 14, more primitive hematopoietic cells, in culture provides strong evidence that the antioxidant capacity of GS-nitroxides improves the interaction between hematopoietic and stromal cells, which is required for maintenance of stem cells in vitro. Adherence of hematopoietic stem cell islands forming cobblestone islands with the bone marrow stromal cell compartment is a parameter previously demonstrated to be associated with resistance to oxidative stress, stable hematopoietic microenvironment, and longevity of hematopoiesis. Thus, the GS-nitroxides demonstrate an in vitro parameter of successful interaction between hematopoietic and stromal cells in an organ culture system. These results confirm and extend those found in vivo showing protection of mouse bone marrow from irradiation (7).
We demonstrated that JP4-039-treated LTBMC-derived stromal cells have an intrinsic up-regulation of multiple gene products, when assayed by RT-PCR. Specifically, we detected increases in Nrf2, Ctgf, Lox, TLR1, COL1a, as well as bromodomain epigenetic reader proteins Brd3, and Brd4. The increase in the adhesion molecule Tlr1 and Col1a were consistent with the flattened epithelial-like morphology of JP4-039 culture-derived stromal cell lines, compared to those from control cultures.
The current data provide further evidence of the safety to the bone marrow and potential therapeutic benefit of GS-nitroxides, specifically JP4-039, in resistance to oxidative stress both from irradiation and in the incubator environment of continuous hematopoiesis in long-term culture.
Acknowledgements
This article was supported by the following grants: NIH R01-CA119927-11 and NIH/NIAID 1U19A168021-06. This project used the UPCI animal facility that is supported in part by award P30CA047904.
- Received February 7, 2014.
- Revision received May 25, 2014.
- Accepted May 26, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
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