James Fortney

Bone marrow stromal cells regulate caspase 3 activity in leukemic cells during chemotherapy

The interaction between leukemic cells and stromal cells of the bone marrow microenvironment has been shown to enhance leukemic cell survival during exposure to chemotherapeutic agents. In the current study we investigated whether association of B lineage acute lymphoblastic leukemic cells with human bone marrow stromal cells altered caspase activation during chemotherapy treatment. Following treatment with Ara-C or VP-16 in vitro, caspase 3 activity in leukemic cells was consistently reduced by co-culture of leukemic cells with human bone marrow stromal cell layers. These observations suggest that the protective effect of the bone marrow microenvironment on leukemic cells may be due, in part, to regulation of caspase 3 activity.

Plasma antioxidant depletion after cardiopulmonary bypass in operations for congenital heart disease.

We describe the use of two in vitro tests to characterize plasma antioxidant capacity at the time of cardiac bypass in operations for congenital heart disease in 30 patients aged 3 days to 16 years (average 4.4 +/- 0.9 years [standard error]). Bypass and crossclamp time, circuit volume, and type of operation were recorded for each patient. First, a test of plasma radical antioxidant power measured chain breaking (secondary) antioxidant capacity of plasma to prevent oxidation of linoleic acid in vitro. Second, overall ability of plasma to prevent lipid peroxidation was assessed by a classic test of plasma inhibition of malondialdehyde formation in a beef brain homogenate. Plasma total radical antioxidant power level at baseline was 0.74 +/- 0.03 mumol/ml plasma, which decreased to 0.15 +/- 0.05 mumol/ml plasma after bypass (p < 0.001) and 0.26 +/- 0.08 mumol/ml plasma with recovery (n = 18, p < 0.001). Analysis of variance of postbypass total radical antioxidant power value showed age (p = 0.0002, r = 0.63) and bypass time (p = 0.009, r = 0.4677) to be significant factors. Pump prime volume in milliliters per kilogram and preoperative hemoglobin value were not significant factors. Beef brain malondialdehyde formation in vitro was limited 92% +/- 3% by normal plasma before operation versus 53% +/- 5% after operation (p < 0.001) and 51% +/- 5% at recovery after arrival in the pediatric intensive care unit (p < 0.001). Analysis of variance of the changes from before to after operation showed age p = 0.0015, r = 0.55) and bypass time (p = 0.033, r = 0.39) to be significant factors. Thus antioxidant capacity of plasma is significantly diminished after cardiopulmonary bypass in children. Young patient age and long duration of cardiopulmonary bypass are identified as factors that correlate positively with depletion of antioxidant capacity with bypass.

Regulation of BAX and BCL‐2 expression in breast cancer cells by chemotherapy

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP‐16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL‐2 expression by VP‐16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen.There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF‐7, MDA‐MB‐435S, and MDA‐MB‐A231 following in vitro treatment with 50–100 μM VP‐16. Elevation of BAX protein expression in the presence of VP‐16 alone did not correlate with reduced viability or induction of apoptosis in MCF‐7, MDA‐MB‐435S, or MDA‐MB‐A231. VP‐16 did effectively block the breast cancer cell lines evaluated (MCF‐7 and MDA‐MB‐435S) at G2/M phase of the cell cycle, confirming activity of the drug in vitro. MCF‐7 and MDA‐MB‐435S cells that were pre‐treated with VP‐16 and subsequently exposed to 1.0–12.0 μg/m1 4‐hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP‐16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP‐16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL‐2 expression.These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.

Activation of Transforming Growth Factor-β1/p38/Smad3 Signaling in Stromal Cells Requires Reactive Oxygen Species–Mediated MMP-2 Activity During Bone Marrow Damage

Dose‐escalated chemotherapy has proven utility in a variety of treatment settings, including preparative regimens before bone marrow or hematopoietic stem cell transplantation. However, the potential damage imposed by aggressive regimens on the marrow microenvironment warrants further investigation. In the present study, we tested the hypothesis that dose‐escalated chemotherapy, with etoposide as a model chemotherapeutic agent, activates the transforming growth factor beta‐1 (TGF‐β1) signaling pathway in bone marrow stromal cells. After high‐dose etoposide exposure in vitro, Smad3 protein was phosphorylated in a time‐and dose‐dependent manner in marrow‐derived stromal cells, coincident with the release of active and latent TGF‐β1 from the extracellular matrix. Phosphorylation was modulated by p38 kinase, with translocation of Smad3 from the cytoplasm to the nucleus subsequent to its phosphorylation. Etoposide induced activation of TGF‐β1 followed the generation of reactive oxygen species and required matrix metalloproteinase‐2 (MMP‐2) protein availability. Chemotherapy effects were diminished in MMP‐2−/− knockout stromal cells and TGF‐β1 knockdown small interfering RNA–transfected stromal cells, in which phosphorylation of Smad3 was negligible after etoposide exposure. Stable transfection of a human MMP‐2 cDNA into bone marrow stromal cells resulted in elevated phosphorylation of Smad3 during chemotherapy. These data suggest TGF‐β1/p38/Smad3 signaling cascades are activated in bone marrow stromal cells after dose‐escalated chemotherapy and may contribute to chemotherapy‐induced alterations of the marrow microenvironment.

Cellular elements of the subarachnoid space promote ALL survival during chemotherapy

CNS infiltration by leukemic cells remains a problematic disease manifestation of acute lymphoblastic leukemia (ALL). Prophylactic regimens for CNS leukemia including intrathecal chemotherapeutics have decreased CNS involvement in ALL, but are not without toxicities. Using co-culture models, we show that astrocytes, choroid plexus epithelial cells, and meningeal cells protect ALL cells from chemotherapy-induced cell death using drugs included in prophylactic regimens-cytarabine, dexamethasone, and methotrexate. Understanding how ALL cells survive in the CNS remains invaluable for designing strategies to prevent CNS leukemia and minimizing the need for treatment in this sensitive anatomical site where treatment-induced toxicity is of significant concern.

VE-cadherin Regulates Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Sensitivity to Apoptosis

The mechanisms by which the bone marrow microenvironment regulates tumor cell survival are diverse. This study describes the novel observation that in addition to Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cell lines, primary patient cells also express Hypoxia Inducible Factor-2α (HIF-2α) and Vascular Endothelial Cadherin (VE-cadherin), which are regulated by Abl kinase. Tumor expression of the classical endothelial protein, VE-cadherin, has been associated with aggressive phenotype and poor prognosis in other models, but has not been investigated in hematopoietic malignancies. Targeted knockdown of VE-cadherin rendered Ph+ ALL cells more susceptible to chemotherapy, even in the presence of bone marrow stromal cell (BMSC) derived survival cues. Pre-treatment of Ph+ ALL cells with ADH100191, a VE-cadherin antagonist, resulted in increased apoptosis during in vitro chemotherapy exposure. Consistent with a role for VE-cadherin in modulation of leukemia cell viability, lentiviral-mediated expression of VE-cadherin in Ph− ALL cells resulted in increased resistance to treatment-induced apoptosis. These observations suggest a novel role for VE-cadherin in modulation of chemoresistance in Ph+ ALL.

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts.

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.

Chemotherapy induces bcl-2 cleavage in lymphoid leukemic cell lines.

Bcl-2 is the major anti-apoptotic protein evaluated in studies aimed at understanding programmed cell death. Recent work suggests that the biological activity of Bcl-2 is modulated by proteolytic cleavage, with a 23 kDa cleaved Bcl-2 product having pro-apoptotic activity. In the current study we evaluated the effect of chemotherapy on Bcl-2 cleavage in B lineage leukemic cell lines. JM-1, SUP-B15 and RS4 leukemic cell lines cleaved Bcl-2 to its 23 kDa form when exposed to the chemotherapeutic agents 1- β - d -arabinofuranosyl-cytosine (Ara-C) or etoposide (VP-16). Chemotherapy induced Bcl-2 cleavage was blunted by inhibition of caspase activity. Co-culture of leukemic cells with bone marrow stromal cells during chemotherapy exposure resulted in reduced levels of 23 kDa Bcl-2 protein. These observations suggest that the bone marrow microenvironment may contribute to maintenance of residual leukemic disease during treatment by reducing generation of pro-apoptotic 23 kDa Bcl-2.

Bone Marrow Osteoblast Damage by Chemotherapeutic Agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.

Alteration of nuclear factor-κB (NF-κB) expression in bone marrow stromal cells treated with etoposide

Abstract Bone marrow stromal cells are an essential regulatory component in the hematopoietic microenvironment. Regulation of hematopoietic cell development is mediated, in part, through interaction of progenitor cells with stromal cell vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 expression has been shown to be driven primarily by binding of nuclear factor-κB (NF-κB) to two consensus binding sites in the promoter region. In this study, we show that down-regulation of VCAM-1 by the chemotherapeutic agent etoposide (VP-16) is associated with altered cellular localization of NF-κB. We demonstrated that VCAM-1 was diminished at the transcriptional level following treatment of stromal cells with VP-16, without alteration of VCAM-1 stability. Culture of bone marrow stromal cells in VP-16 resulted in reduced nuclear RelA (p65), a modest increase in nuclear NF-κB1 (p50), and reduced NF-κB binding to its DNA consensus sequence. Total levels of the NF-κB inhibitor Iκ-Bα were reduced during exposure to VP-16. Following removal of VP-16 from the culture, p65 and p50 nuclear profiles approximated those of untreated stromal cells, and VCAM-1 protein expression was restored. The current study indicates that NF-κB is a target molecule that is responsive to VP-16-induced damage in bone marrow stromal cells. As the primary transcription factor that promotes VCAM-1 expression, the observed changes in p65 and p50 cellular localization during treatment have a direct consequence for stromal cell function. The myriad of genes regulated by NF-κB, including both adhesion molecules and cytokines that contribute to stromal cell function, make chemotherapy-induced disruption of NF-κB biologically significant. Alterations in NF-κB activity may provide one measure by which the effects of aggressive treatment strategies on the bone marrow microenvironment can be evaluated.