News and Publications

Normal and Leukemic Hematopoiesis

J. Yu, D.P. Dialynas, L.E. Shao, M.J. Lee, A. Galaviz

ACTIVIN A IN HEMATOPOIESIS AND INFLAMMATION

Activin A, a member of the transforming growth factor-ß superfamily, plays roles in differentiation and development, including hematopoiesis. Research by us and by others indicated that activin A potentiates the proliferation and differentiation of human erythroid progenitor cells. Other studies revealed that activin A can expand the erythroid progenitors in cell cultures in the absence of erythropoietin, if a stem cell factor is present. Furthermore, dissuccinimidyl suberate chemically cross-linked radiolabeled activin A to 2 cell-surface binding proteins in purified erythroid progenitors and K562 cells. Current studies suggest that the potentiation effect of activin A in the proliferation of erythroid progenitor cells involves the gp130 signal transduction pathway.

Our investigations also indicated that expression of activin A in human bone marrow stromal cells and monocytes is highly regulated by inflammatory cytokines and glucocorticoids. We identified a transcription start site at 212 nucleotides upstream from the translation start codon. We also found that a 58-nucleotide upstream sequence has promoter activity and that increased expression of activin A requires a positive element consisting of an additional 71 nucleotides of the upstream region.

However, activin A is also a cytokine with multiple functions that are not fully determined. Our studies of synoviocytes and chondrocytes in cultures indicated that production of activin A in these cells was also stimulated by proinflammatory cytokines. Other results showed that patients with rheumatoid arthritis and gout had higher concentrations of activin A in the synovial fluid than did patients with osteoarthritis. Further studies revealed that several IL-6--induced activities that lead to systemic inflammatory responses were suppressed by activin A. These data suggest additional facets of inflammation-related activities for activin A.

In addition, we used several human macrophage lines developed in the laboratory to systematically analyze HLA-DR--driven macrophage activation. In particular, we characterized cytokine regulation of macrophage HLA class II signaling and the consequential effector output. This work led directly to a novel model for the potential contribution of HLA-DR--restricted macrophage activation to the pathogenesis of rheumatoid arthritis. The model, which considers an alternative mechanistic basis for the association of HLA-DR with rheumatoid arthritis, can directly accommodate a number of clinical observations that are otherwise difficult to reconcile. The model also suggests both a novel screen for the putative peptide or peptides associated with rheumatoid arthritis and a number of novel targets for therapeutic intervention.

NEGATIVE REGULATION OF HEMATOPOIESIS BY CYTOKINES

It is thought that hematopoiesis is regulated by both positive and negative signals derived from the microenvironment of the bone marrow, which includes macrophages. The proteins that mediate negative regulation and their mechanism of action are an area of active investigation. We functionally and biochemically characterized a suppressor of early hematopoietic progenitors, designated NRH, isolated from the recently established human macrophage line 2MAC. The mechanism of action of NRH suppression appears to involve a marked decrease in the cycling of early progenitor cells. NRH activity was reversible and corresponded to a heparin-binding glycoprotein with a molecular weight of about 20 kD.

We developed a procedure for the rapid isolation of highly purified NRH from 2MAC conditioned medium. According to a number of functional and biochemical criteria, NRH appears to be a novel macrophage-derived negative regulator of hematopoiesis that may be useful in certain clinical settings as a chemoprotectant of primitive hematopoietic cells.

THIOREDOXIN IN PRIMARY ACUTE LYMPHOBLASTIC LEUKEMIA

Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children. Our studies and those of others suggest that thioredoxin may be an oncoprotein for human cancers and support the thioredoxin redox system as a rational target for selective anticancer therapy. Thioredoxin induces expression of the high-affinity IL-2 receptor -chain and exerts redox control over transcription factors such as NF-B, TFIIIC, BZLF1, and Ref-1. Overexpression of thioredoxin in cells suppresses apoptosis and is associated with drug sensitivity to chemotherapy.

Through the nationwide Pediatric Oncology Group, we have access to samples of primary leukemia cells collected from children who are treated uniformly. We are measuring the expression of thioredoxin in primary ALL cells and are testing the hypothesis that high expression of thioredoxin may be related to less desirable clinical features and outcomes. In collaboration with G. Powis, University of Arizona, we determined the in vitro sensitivity of primary ALL cells to inhibitors of thioredoxin, such as 1-methylpropyl-2-imidazolyl disulfide and other related compounds. In addition, we will examine the in vivo effect of thioredoxin inhibitors on the growth of leukemia cells in our human ALL preclinical model.

MOUSE MODELS OF LEUKEMIC HEMATOPOIESIS

We developed an experimental strategy for generating a murine model of human leukemias and other malignant tumors. Using a novel preconditioning procedure, we reconstituted nonobese mice with severe combined immunodeficiency disease with primary tumor cells obtained from patients with leukemia, including T-cell ALL (T-ALL). The progression and dissemination of the human leukemia cells in these mice mimicked the clinical features of patients, but the rate and the overall success of engraftment were substantially better than previously reported. Using this murine model, in collaboration with D.J. Flavell, University of Southampton, England, we are investigating the efficacy of the newly developed anti-CD5, anti-CD7, and anti-CD38 immunotoxins, conjugated with saporin, for the treatment of human T-ALL. In addition, in collaboration with C.J. Carrera, University of California, San Diego, we will analyze the efficacy of l-alanosine, especially in mice reconstituted with cells from T-ALL patients with methylthioadenosine phosphorylase--deficient malignant tumors.

The ability to engraft leukemic cells or malignant tumors directly from patients into mice could be valuable for predicting the clinical course of the neoplastic disease, detecting residual tumor cells, and developing individualized therapeutic strategies. This model will also be used to study tumor biology, including the in vivo homing, engraftment, progression, and metastasis of the tumor and the disease heterogeneity of leukemia and other malignant neoplasms.

PUBLICATIONS

Dialynas, D.P., Tan, P.C., Yu, J. Cytokine modulatable signalling through macrophage HLA class II: 1. IFN up-regulates the efficiency of Ca²⁺ mobilization in response to ligation of macrophage HLA-DP. J. Interferon Cytokine Res. 17:671, 1997.

Dialynas, D.P., Yu, J. A model for the potential contribution of HLA-DA-restricted macrophage activation to the pathogenesis of rheumatoid arthritis. Arthritis Rheum. 41:1515, 1998.

Diccianni, M.B., Batova, A., Yu, J., Vu, T., Pullen, J., Amylon, M., Pollock, B., Yu, A.L. Shortened survival after relapse in T-cell acute lymphoblastic leukemia patients with p16/p15 deletions. Leuk. Res. 21:549, 1997.

Dolter, K.E., Palyash, J.C., Shao, L.-E., Yu, J. Analysis of activin A gene expression in human bone marrow stromal cells. J. Cell. Biochem. 70:8, 1998.

Shao, L.-E., Frigon, N.L., Jr., Yu, A., Palyash, J., Yu, J. Contrasting effects of inflammatory cytokines and glucocorticoids on the production of activin A in human marrow stromal cells and their implications. Cytokine 10:227, 1998.

Yu, E.W., Dolter, K.E., Yu, J. Suppression of IL-6 biological activities by activin A and implications for inflammatory arthropathies. Clin. Exp. Immunol. 112:126, 1998.

Yu, J. Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells. Blood Cells Mol. Dis. 24:166, 1998.

Yu, J., Dolter, K.E. Production of activin A and its roles in inflammation and hematopoiesis. Cytokines Cell. Mol. Ther. 3:169, 1997.