Oncogenic Role for the Lck/ZAP70/PLCG2 Signaling Pathway in Pre-B-ALL Pathogenesis

Abstract Although the treatment and prognosis of patients with pediatric acute lymphoblastic leukemia (ALL) have improved during the last decades, there is still a clinical need for more effective/selective and less toxic therapies. To address this, we have interrogated various signaling pathways in human ALL cells and mouse strains that express E2A-PBX1, which is present in 5-7% of pediatric ALL. Phospho-flow analysis revealed basal hyper-phosphorylation levels of PLCγ2 in mouse E2A-PBX1 leukemias, consistent with hyper-activation of upstream signaling pathways. Efficient shRNA-mediated depletion of PLCγ2 reduced colony formation of mouse E2A-PBX1+ leukemias in vitro and increased disease-free survival after secondary bone marrow transplantation in vivo. Furthermore, PLCγ2-depleted human ALL cell lines including E2A-PBX1+ cells, showed reduced proliferation. These data suggest a pathogenic role of hyperactivated PLCγ2 in pre-B-ALL. Bioinformatics analysis of E2A-PBX1 target genes in human ALLs revealed an enrichment of B- and T-cell activation pathways, which include the SRC-family kinase LCK and the cytoplasmic kinase ZAP70, upstream of PLCγ2. Comparative analyses of global transcriptional profiles in human primary and mouse leukemias and preleukemias induced by the E2A-PBX1 oncogene identified the signaling kinase ZAP70 as one of the earliest and most consistently up-regulated genes in E2A-PBX1 leukemias. Using a candidate gene approach, we identified LCK with increased expression levels in E2A-PBX1 leukemia cells compared to normal B-cell progenitors. Mouse and human E2A-PBX1 leukemia cells were dependent on the E2A-PBX1 target genes ZAP70 and LCK for proliferation and survival as confirmed by shRNA knock-down experiments. Hence, efficient depletion of these genes resulted in a decrease of phosphorylated PLCγ2, suggesting therapeutic targets in E2A-PBX1 leukemias. Combined suppression of ZAP70 and LCK using double-shRNA experiments showed an additive effect on inhibition of cell proliferation and decrease of phosphorylated PLCγ2. These results provide a rationale for combination therapy to block this hyper-activated signaling pathway at different levels. Several small molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC-family kinase inhibitors including dasatinib were most effective in reducing phosphorylation of PLCγ2 and inhibiting cell proliferation. Furthermore, dasatinib showed promising preclinical efficacy in vitro in colony forming assays and in vivo after secondary bone marrow transplantation of leukemias. In summary, our studies demonstrate that the proliferation and survival of E2A-PBX1 leukemias are dependent on PLCγ2 and upstream signaling pathways, which are suitable for pharmacological inhibition. Disclosures No relevant conflicts of interest to declare.

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HPV-18 E6 Oncoprotein and Its Spliced Isoform E6*I Regulate the Wnt/β-Catenin Cell Signaling Pathway through the TCF-4 Transcriptional Factor

International Journal of Molecular Sciences ◽

10.3390/ijms19103153 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3153 ◽

Cited By ~ 6

Author(s):

J. Muñoz-Bello ◽

Leslie Olmedo-Nieva ◽

Leonardo Castro-Muñoz ◽

Joaquín Manzo-Merino ◽

Adriana Contreras-Paredes ◽

...

Keyword(s):

Cervical Cancer ◽

Cell Proliferation ◽

Signaling Pathway ◽

Transcriptional Activation ◽

Target Genes ◽

Promote Cell Proliferation ◽

E6 And E7 ◽

Hpv 18

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/β-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/β-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and β-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with β-catenin to promote cell proliferation.

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Persistence of myeloid progenitor cells expressing BCR-ABL mRNA after allogeneic bone marrow transplantation for chronic myelogenous leukemia

Blood ◽

10.1182/blood.v84.7.2109.bloodjournal8472109 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2109-2114

Author(s):

G Pichert ◽

EP Alyea ◽

RJ Soiffer ◽

DC Roy ◽

J Ritz

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Progenitor Cell ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone Marrow ◽

Myeloid Differentiation ◽

Allogeneic Bone

Previous studies have shown that tumor-specific bcr-abl mRNA can often be detected by polymerase chain reaction. (PCR) for months to years after allogeneic bone marrow transplantation (BMT) for chronic myelocytic leukemia (CML). Nevertheless, the presence of bcr-abl mRNA by itself does not invariably predict for clinical relapse post-BMT. This has led to the hypothesis that bcr-abl mRNA might be expressed in cells that have lost either proliferative or myeloid differentiation potential. To directly characterize the cells detected by PCR in patients with CML after allogeneic BMT, we first identified five individuals in whom PCR-positive cells could be detected at multiple times post-BMT. Bone marrow samples from these individuals were cultured in vitro and single erythroid, granulocytic, and macrophage colonies, each containing 50 to 100 cells, were examined for the presence of bcr-abl mRNA by PCR. PCR-positive myeloid colonies could be detected in four of five individuals in marrow samples obtained 5 to 56 months post-BMT. Overall, 7 of 135 progenitor cell colonies (5.2%) were found to be PCR-positive. The expression of bcr-abl mRNA appeared to be equally distributed among committed erythroid, macrophage, and granulocyte progenitors. These patients have now been followed-up for an additional 20 to 33 months from the time of progenitor cell PCR analysis but only one of these individuals has been found to have cytogenetic evidence of recurrent Ph+ cells. These results show that long-term persistence of PCR-detectable bcr-abl mRNA after allogeneic BMT can be caused by the persistence of CML-derived clonogenic myeloid precursors that have survived the BMT preparative regimen. These cells continue to have both proliferative and myeloid differentiation capacity in vitro. Nevertheless, these PCR-positive cells do not appear to either expand or differentiate in vivo for prolonged periods, suggesting the presence of mechanisms for suppression of residual clonogenic leukemia cells in vivo.

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Gamma-interferon and tumor necrosis factor production after bone marrow transplantation is augmented by exposure to marrow fibroblasts infected with cytomegalovirus

Blood ◽

10.1182/blood.v76.5.1046.1046 ◽

1990 ◽

Vol 76 (5) ◽

pp. 1046-1053 ◽

Cited By ~ 10

Author(s):

AS Duncombe ◽

A Meager ◽

HG Prentice ◽

JE Grundy ◽

HE Heslop ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Tumor Necrosis Factor ◽

Marrow Transplantation ◽

Tumor Necrosis ◽

Gamma Interferon ◽

Cmv Infection ◽

Necrosis Factor

Abstract After bone marrow transplantation (BMT), mortality from viral infections such as cytomegalovirus (CMV) remains high. Gamma-Interferon (gamma IFN) and tumor necrosis factor (TNF) are produced constitutively after BMT and have anti-viral properties. To study the effects of these cytokines on CMV interaction with host cells, we have used patient marrow fibroblasts since marrow stroma is a target for CMV infection correlating with myelosuppression in vivo. Both gamma IFN and TNF are constitutively produced by recipient CD3+ and CD16+ lymphocytes, but not by their marrow fibroblasts. Secretion by peripheral blood mononuclear cells is increased if they are cultured with host fibroblasts infected with CMV in vitro and the levels of gamma IFN and TNF produced are within the range that protects fresh fibroblasts from CMV infection. Constitutive secretion of cytokines by lymphocytes declines by 8 weeks after BMT, a time when the risk of CMV disease increases sharply. The in vitro phenomenon that we have described needs to be evaluated in correlative studies on individual BMT recipients to determine whether such a cytokine-mediated defense mechanism against CMV may operate in vivo.

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CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v110.11.1224.1224 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1224-1224

Author(s):

Jerry C. Cheng ◽

Dejah Judelson ◽

Kentaro Kinjo ◽

Jenny Chang ◽

Elliot Landaw ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Mouse Bone Marrow ◽

T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

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Human Mesenchymal Stem Cells Attenuate Graft-Versus-Host Disease and Maintain Graft-Versus-Leukemia in Murine Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v118.21.1907.1907 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1907-1907 ◽

Cited By ~ 1

Author(s):

Jeffery J Auletta ◽

Saada Eid ◽

Matthew Keller ◽

Leland Metheny ◽

Rocio Guardia-Wolff ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Graft Versus Host Disease ◽

Marrow Transplantation ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Graft Versus Host ◽

Graft Versus Leukemia

Abstract Abstract 1907 Defining in vivo effects and biodistribution of human bone marrow-derived mesenchymal stem cell (hMSCs) following allogeneic bone marrow transplantation (alloBMT) could impact the clinical utility of MSC therapy for the prevention and treatment of graft-versus-host disease (GvHD). Using an established model of murine alloBMT, we defined hMSC effects on GvHD and graft-versus-leukemia (GvL) activity. We first studied whether hMSC could modulate in vitro murine T-cell (TC) alloreactivity in mixed leukocyte cultures (MLCs). Specifically, hMSCs added to MLCs significantly reduced TC proliferation in a concentration-dependent manner distinct from human fibroblasts. In contrast to MLC cultures alone, MLCs containing hMSCs had significant reduction in TNFα, IFNγ, and IL-10 levels and higher levels of PGE2 and TGFβ1. Modulation in the inflammatory milieu was associated with changes in TC phenotypes, including more naïve and less activated TC surface marker expression (CD62L+CD69−) and the induction of CD4+CD25+FoxP3+ T-regulatory cells. To determine whether hMSCs could modulate in vivo mTC alloreactivity, irradiated recipient B6D2F1 (H-2bxd) mice were transplanted with allogeneic C57BL/6 (H-2b) BM and purified splenic TCs (B6→B6D2F1) and then were tail-vein injected with hMSC infusions (1 million per injection) on days one and four post-transplant. Syngeneic transplant recipients (B6D2F1→B6D2F1) were used as controls. hMSC-treated alloBMT mice had significantly prolonged survival and improved clinical GvHD scores, reduced splenic TC expansion and TNFα and IFNγ-producing TCs, and lower circulating TNFα and IFNγ levels versus untreated alloBMT mice. Bioluminescence imaging showed redistribution of labeled hMSCs from the lungs to abdominal organs within 72 hours following infusion. Importantly, GvHD target tissues (small and large bowel and liver) harvested from hMSC-treated alloBMT mice had significantly lower GvHD pathology scores than untreated alloBMT mice. We next determined the effects of hMSCs on GvL activity using the murine mastocytoma cell line, P815 (H-2d). TCs co-cultured with hMSCs maintained potent in vitro cytotoxic T-lymphocyte (CTL) activity comparable to untreated control CTLs. After challenge with P815 tumor cells, hMSCs-treated alloBMT mice had less severe GvHD, eradication of tumor burden, and improved leukemia-free survival compared to alloBMT control mice. Lastly, indomethacin (IM) added to MLC-hMSC co-cultures significantly reversed attenuation in both murine TC alloreactivity and surface activation expression. In addition, IM administered to hMSC-treated alloBMT mice reversed hMSC-associated survival advantage, suggesting that PGE2 in part mediates hMSC immunomodulatory effects. Together, our results show that hMSC infusions effectively attenuate GvHD and maintain GvL potency in alloBMT mice and reveal potential biomarkers and mechanisms of action underlying hMSC effects. Disclosures: Solchaga: Bimemetic Therapeutics: Employment. Cooke:Amgen: Provides experimental drug and central pharmacy support for 2 trials for which I am Co-PI.

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Susceptibility of AML to Chloroquine Therapy Is Independent of Autophagy

Blood ◽

10.1182/blood.v126.23.1262.1262 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1262-1262 ◽

Cited By ~ 1

Author(s):

Xiaoyi Chen ◽

Jason Clark ◽

Jun-lin Guan ◽

Ashish R Kumar ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Specific Targeting ◽

Xenograft Mice ◽

Autophagy Activity ◽

Marrow Cells

Abstract Introduction: autophagy is a self-recycling and "waste disposal" process that maintains cellular homeostasis. Recently, the autophagy mechanism has evolved as a therapeutic target in cancer treatment due to commonly seen high autophagy activity in cancer cells and its potential role in chemoresistance. To date, inhibition of autophagy has shown therapeutic benefits in several types of solid tumors. However, whether autophagy can be a potentially effective target in AML therapy remains unclear. Here we have used autophagy gene targeted mouse models and drug inhibitors to examine the potential benefits and limitations of autophagy targeting in the treatment of AML. Methods: MLL-AF9 (MA9) oncogene transduced Atg5f/fCreER and Atg5f/fMxCre Lin- mouse bone marrow cells were used for in vitro and in vivo experiments, respectively. Autophagy activity was determined by biochemical Western Blotting and immunofluorescent microscopy against LC3 and electron microscopy (EM). Deletion of FIP200, a gene that is indispensable for both canonical and alternative autophagy pathways, was carried out similarly to further confirm the effect of autophagy-specific targeting on MA9 leukemia. Chloroquine (CQ), a late stage autophagy and lysosome inhibitor, was used at 10μM to 25μM dosages in vitro. Combinatory effects of CQ with chemotherapy, including Doxorubicin (DA) and Cytarabine (AraC), were also analyzed. CQ was also administered to mice through i.p., at 50mg/Kg, bid X 4 days. Leukemia burden, cell survival and CBC counts were analyzed after drug treatment. Results: Primary and clonal MA9 leukemia cells showed a significantly higher level of autophagy flux than normal bone marrow cells. As expected, Atg5-/- MA9 cells showed defective LC3II formation and higher p62 accumulation upon CQ treatment. However, Atg5 deletion did not cause detectable defects in proliferation or survival, or altered ROS or mitochondria levels in leukemia cells. Surprisingly, Atg5-/- MA9 leukemia cells showed reduced sensitivity to either DA or AraC treatment. Transplantation experiments showed that Atg5 deletion in vivo did not reduce leukemia burden in the bone marrow or prolong the survival of the leukemic mice, although it decreased WBC counts in peripheral blood. When examined by EM, no obvious ultrastructural difference was observed between Atg5+/+ and Atg5-/- leukemia cells and both could form endolysosomes upon CQ blockage. Although FIP200 deletion in MA9 leukemia cells caused p62 accumulation at the basal state, similar functional effects were seen as in the case of Atg5 deletion. When compared to wild type controls, FIP200-/- MA9 leukemia cells did not show any proliferation or survival disadvantage, changes in ROS accumulation or mitochondrial level. FIP200 deletion also failed to sensitize leukemia cells to chemotherapy. Finally, CQ independently suppressed leukemia cell proliferation and induced apoptosis, but it did not distinguish Atg5+/+ vs Atg5-/- or FIP200+/+ vs FIP200-/- MA9 leukemia cells in sensitivity. CQ also showed a combinatory effect with DA or AraC in inhibiting MA9 cell proliferation. Treatment of MA9 leukemia xenograft mice with CQ greatly improved anemia in the mice (P<0.01), and we are currently examining the potential effects of CQ on survival of the leukemia bearing mice. Conclusions: MA9 leukemia cells contain a high basal autophagy activity. However, autophagy-specific targeting, either through FIP200-deletion which abolishes autophagy initiation, or via Atg5-deletion which prevents autophagosome membrane elongation, does not affect the survival and proliferation of MA9 leukemia cells nor does it prolong survival of MA9 leukemia xenograft mice. These results suggest that targeting autophagy alone will unlikely produce therapeutic benefit in AML. Loss of Atg5or FIP200 does not sensitize leukemia cells to chemotherapy, further reducing the potential value of targeting autophagy in a combinatory chemotherapy scheme. Mechanistically, neither Atg5 nor FIP200 is required for proper endolysosome-formation or lysosomal degradation in leukemia cells. While CQ displayed an apparent anti-leukemic effect, it acts through an autophagy independent pathway. CQ has a combinatory effect with conventional chemotherapy drugs and may be a useful treatment regimen for MA9 mediated and other types of AML. Disclosures No relevant conflicts of interest to declare.

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Gamma-interferon and tumor necrosis factor production after bone marrow transplantation is augmented by exposure to marrow fibroblasts infected with cytomegalovirus

Blood ◽

10.1182/blood.v76.5.1046.bloodjournal7651046 ◽

1990 ◽

Vol 76 (5) ◽

pp. 1046-1053

Author(s):

AS Duncombe ◽

A Meager ◽

HG Prentice ◽

JE Grundy ◽

HE Heslop ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Tumor Necrosis Factor ◽

Marrow Transplantation ◽

Tumor Necrosis ◽

Gamma Interferon ◽

Cmv Infection ◽

Necrosis Factor

After bone marrow transplantation (BMT), mortality from viral infections such as cytomegalovirus (CMV) remains high. Gamma-Interferon (gamma IFN) and tumor necrosis factor (TNF) are produced constitutively after BMT and have anti-viral properties. To study the effects of these cytokines on CMV interaction with host cells, we have used patient marrow fibroblasts since marrow stroma is a target for CMV infection correlating with myelosuppression in vivo. Both gamma IFN and TNF are constitutively produced by recipient CD3+ and CD16+ lymphocytes, but not by their marrow fibroblasts. Secretion by peripheral blood mononuclear cells is increased if they are cultured with host fibroblasts infected with CMV in vitro and the levels of gamma IFN and TNF produced are within the range that protects fresh fibroblasts from CMV infection. Constitutive secretion of cytokines by lymphocytes declines by 8 weeks after BMT, a time when the risk of CMV disease increases sharply. The in vitro phenomenon that we have described needs to be evaluated in correlative studies on individual BMT recipients to determine whether such a cytokine-mediated defense mechanism against CMV may operate in vivo.

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CD6-Depletion in the Dog Model: New Insights into Chimerism and Tolerance.

Blood ◽

10.1182/blood.v110.11.2173.2173 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2173-2173

Author(s):

Julia Zorn ◽

Martin Herber ◽

Sabine Schwamberger ◽

Heiko Adler ◽

Hans-Jochem Kolb

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Canine Model ◽

In Vivo Studies ◽

Control Group ◽

Human Antibody ◽

Rabbit Complement

Abstract Adoptive immunotherapy with donor lymphocyte infusion (DLI) following bone marrow transplantation may produce a graft-versus-leukemia (GVL) effect after transplantation of T-cell-depleted bone marrow. However, this method is limited by a high risk of GvHD. We studied the occurrence of graft-versus-host disease (GVHD) and chimerism after DLI at various times after CD6-depleted bone marrow transplantation in a canine model of DLA-homozygous donor and DLA-heterozygous littermate recipient. Using a cross reactive mouse-anti-human antibody which recognizes the canine CD6 antigen, a simple and economical depletion method could be established, which makes use of the ability of the CD6 antibody MT-606 to activate the complement cascade. In vitro experiments using MT-606 and rabbit complement showed the effective depletion of CD6-positive cells whereas hematopoietic progenitor cells remained unaffected at the same time. For in vivo studies, DLA heterozygous recipients with one DLA haplotype being identical to the donor were treated with 10 Gy total body irradiation (TBI) and received CD6-depleted bone marrow of their DLA-homozygous littermate donors. In the control group 7 dogs given unmanipulated marrow died of GVHD within 28 days of transplantation. Two dogs showed sustained engraftment without the occurrence of GvHD after the infusion of 1,8 · 108/kg – 2,0· 108/kg CD6-depleted mononuclear marrow cells. The establishment of tolerance to the bone marrow donor was shown by the transplantation of skin from the donor to the host. Transfusion of donor lymphocytes (1.0 · 108 MNCs/kg body weight) on day 3, 7 or 14 induced GvHD in the host. However, with DLI on day 20, only 2 out of 4 dogs developed GvHD, but DLI always converted mixed into complete chimerism. We conclude from this study that the cross reactive CD6 antibody MT606 effectively depletes T cells in the presence of rabbit complement and induces GVH tolerance without jeopardizing engraftment, and the risk of GVHD after DLI is reduced with time after transplantation.

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Comparative effects of in vivo treatment using interleukin-11 and stem cell factor on reconstitution in mice after bone marrow transplantation

Blood ◽

10.1182/blood.v82.3.1016.1016 ◽

1993 ◽

Vol 82 (3) ◽

pp. 1016-1022 ◽

Cited By ~ 36

Author(s):

XX Du ◽

D Keller ◽

R Maze ◽

DA Williams

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Growth Factor ◽

Marrow Transplantation ◽

Stem Cell Factor ◽

Erythroid Cell ◽

Cell Recovery

Abstract Molecular analysis of the hematopoietic microenvironment (HM) has led to the characterization and molecular cloning of two unique growth factors produced by stromal cells. Interleukin (IL)-11 and stem-cell factor (SCF; steel factor [SF]) have been shown in a variety of in vitro culture systems to stimulate distinct populations of stem, progenitor, and more differentiated cell types. We have analyzed and compared the effects of each growth factor administered to mice undergoing bone marrow transplantation (BMT) after total body irradiation (TBI). We report that IL-11 stimulates platelet and neutrophil recovery, while the main effect of SCF is on erythroid cell recovery in this model. Mice treated with the combination of IL-11 and SCF show increases in all three lineages compared with control mice, without obvious toxicity. In addition, both the type of progenitor- and stem-cell populations stimulated and the anatomic localization of effects seen with each growth factor are distinct. These data in mice suggest that the combination of IL-11 and SCF might be useful in humans undergoing myeloablative therapies.

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Persistence of myeloid progenitor cells expressing BCR-ABL mRNA after allogeneic bone marrow transplantation for chronic myelogenous leukemia

Blood ◽

10.1182/blood.v84.7.2109.2109 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2109-2114 ◽

Cited By ~ 27

Author(s):

G Pichert ◽

EP Alyea ◽

RJ Soiffer ◽

DC Roy ◽

J Ritz

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Progenitor Cell ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone Marrow ◽

Myeloid Differentiation ◽

Allogeneic Bone

Abstract Previous studies have shown that tumor-specific bcr-abl mRNA can often be detected by polymerase chain reaction. (PCR) for months to years after allogeneic bone marrow transplantation (BMT) for chronic myelocytic leukemia (CML). Nevertheless, the presence of bcr-abl mRNA by itself does not invariably predict for clinical relapse post-BMT. This has led to the hypothesis that bcr-abl mRNA might be expressed in cells that have lost either proliferative or myeloid differentiation potential. To directly characterize the cells detected by PCR in patients with CML after allogeneic BMT, we first identified five individuals in whom PCR-positive cells could be detected at multiple times post-BMT. Bone marrow samples from these individuals were cultured in vitro and single erythroid, granulocytic, and macrophage colonies, each containing 50 to 100 cells, were examined for the presence of bcr-abl mRNA by PCR. PCR-positive myeloid colonies could be detected in four of five individuals in marrow samples obtained 5 to 56 months post-BMT. Overall, 7 of 135 progenitor cell colonies (5.2%) were found to be PCR-positive. The expression of bcr-abl mRNA appeared to be equally distributed among committed erythroid, macrophage, and granulocyte progenitors. These patients have now been followed-up for an additional 20 to 33 months from the time of progenitor cell PCR analysis but only one of these individuals has been found to have cytogenetic evidence of recurrent Ph+ cells. These results show that long-term persistence of PCR-detectable bcr-abl mRNA after allogeneic BMT can be caused by the persistence of CML-derived clonogenic myeloid precursors that have survived the BMT preparative regimen. These cells continue to have both proliferative and myeloid differentiation capacity in vitro. Nevertheless, these PCR-positive cells do not appear to either expand or differentiate in vivo for prolonged periods, suggesting the presence of mechanisms for suppression of residual clonogenic leukemia cells in vivo.

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