John Yu

Induced expression of the new cytokine, activin A, in human monocytes: inhibition by glucocorticoids and retinoic acid.

The capacity of recombinant human granulocyte–macrophage colony‐stimulating factor (GM‐CSF), glucocorticoids or all‐trans‐retinoic acid to modulate production of activin A by human monocytes was studied. It was shown that GM‐CSF stimulated monocytes to accumulate activin A RNA after as few as 4 hr of incubation, reaching a peak of stimulation at approximately 16 hr of incubation. The activin A transcripts accumulated in the monocytes after stimulation with only 5 U/ml of GM‐CSF and reached a maximum plateau level of expression between 25 and 50 U/ml of GM‐CSF. Biologically active activin A molecules were detected in the conditioned media by a bioassay, performed both in the absence and presence of a neutralizing antiserum for activin A. Accumulation of bioactive activin A in conditioned medium of monocyte cultures was detected after 24 hr of incubation with GM‐CSF and high levels of activin A were maintained for 72 hr. The production of the dimeric βAβA in these monocytes was further confirmed by a sandwich enzyme‐linked immunosorbent assay (ELISA) specific for activin A. In contrast to the stimulatory effect of GM‐CSF, hydrocortisone, dexamethasone or all‐trans‐retinoic acid at 1 × 10−7 to 1 × 10−5 m inhibited the constitutive expression of activin A and greatly suppressed the GM‐CSF‐stimulated production. Thus, the expression of activin A is modulated in monocytes by different agents. These observations may imply new roles for activin A at sites of inflammation where monocytes accumulate.

Expression of activin A in inflammatory arthropathies.

The findings that bone marrow fibroblastoid stromal cells are important for activin A production prompted our investigation of activin A expression in fibroblast-like synoviocytes in joint capsule in this and previous studies. In the proliferative reactive synovial membrane obtained from rheumatoid arthritis patients, activin A is detected prominently in the fibroblastoid synovial cells, as well as in the smooth muscle and the endothelial layer of the arteries in these vascularized proliferative tissues. The concentration of activin A in the rheumatoid arthritis synovial fluid was 33.6+/-5.0 ng/ml, much higher than the activin A content of osteoarthritis fluid (10.0+/-1.1 ng/ml). Furthermore, our previous studies also showed that inflammatory cytokines, such as interleukin (IL)-1, transforming growth factor (TGF)-beta, interferon (IFN)-gamma, IL-8, and IL-10 markedly enhance the expression of activin A mRNA in synoviocytes. These findings are consistent with our studies in regard to the regulatory control of activin A production in bone marrow stroma and monocytes. In addition, the relationship of activin A to IL-6-induced biological activities in various cell types was also investigated. Although activin A has not been directly associated with inflammatory processes, future studies are needed to investigate its production in response to the accumulated levels of inflammatory cytokines in the synovium of the patients, as well as the quantitative differences in activin A concentrations in many patients with other inflammatory diseases.

Shortened survival after relapse in T-cell acute lymphoblastic leukemia patients with p16/p15 deletions

p16 Alterations were detected in > 60% of 103 primary T-ALL samples. In paired diagnosis-relapse patient samples, 80% of the relapse samples with p16 deletion were deleted at diagnosis. When p16 was homozygously deleted, p15 gene alterations were found in 72% of the diagnosis T-ALL patient samples, increasing significantly to 100% at relapse. Alterations of p18 were not detected. No clinical significance of p15/p16 gene deletion in diagnosis T-ALL was found with respect to white blood cell (WBC) count, incidence of mediastinal mass, rate of relapse, duration of first remission or event-free survival. In relapse T-ALL, however, patients with p16 deletion experienced a significantly shorter duration of post-relapse survival, demonstrating that p16 deletion is clinically significant in T-ALL.

Thioredoxin-Related Regulation of NO/NOS Activities

Abstract: The role of regulation of nitric oxide synthase (NOS) activity in mitigating oxidative stress in neonatal lungs and contributing to pulmonary vasodilation at birth is still unclear. Furthermore, it is known that, depending on interactions between the individual components of the mitogen‐activated protein kinase (MAPK) signaling cascades, many biological consequences, including apoptosis, are initiated. Although the importance of nitric oxide (NO) in apoptosis is controversial and likely depends on NO concentrations and cell types, this highly reactive free radical can activate the p38 MAPK signal cascade. Recent studies have suggested that thioredoxin may play an important role as an effector for some of these functions. Thioredoxin is a major redox protein for many enzymes/transcription factors and is involved in cellular functions, such as viability, activation, and proliferation. In addition to its redox regulation, thioredoxin binds directly to the apoptosis signal‐regulating kinase 1 (ASK1), thus inhibiting the activation of stress‐induced MAPK signaling cascades that lead to apoptosis. Furthermore, NO produced from newly induced neuronal NOS was reported to induce expression of thioredoxin and several other genes for preconditioning‐induced neuroprotection. Moreover, although exposure of endothelial cells to NO decreases NOS activity, this inhibition was shown to be reversed by thioredoxin. Finally, the correlation of expression of thioredoxin with endothelial NOS activity seems to suggest an important role played by this protein in perinatal changes of pulmonary artery functions. Therefore, thioredoxin may participate in the regulation of NOS activity and be involved in NO functions via multiple mechanisms.

Detection of Functional and Dimeric Activin A in Human Marrow Microenvironment

Activin A, which was initially recognized as a gonadal protein, was implicated in the modulation of erythropoiesis through a paracrine control in the bone marrow microenvironment. Present studies demonstrate that, in contrast to T lymphocytes and cultured skin fibroblasts, human marrow stromal cells produce a functional and dimeric beta A beta A molecule (i.e., activin A). RT-PCR further indicates that both alpha and beta A mRNAs of inhibin A/activin A are produced in human stromal cells. The level of beta A subunit mRNAs, however, is in large excess over that of alpha subunit mRNAs, suggesting the predominant production of beta A beta A dimers, as well as some inhibin A (alpha beta A). It should be noted, however, that the beta A subunit can form dimeric proteins other than activin A, such as activin AB (beta A beta B) and inhibin A (alpha beta A). Hence, the presence of the beta A subunit may not necessarily indicate the production of the activin A molecule in any tissue. Therefore, a special quantitative sandwich ELISA assay specific for the dimeric beta A beta A molecule was developed for the measurement of activin A. With this assay, production of activin A in marrow stromal cells is found to be greatly enhanced by cytokines and inflammatory mediators such as TNF-alpha, IL-1 alpha, and lipopolysaccharide. These studies thus suggest that inflammatory cytokines are the inducers for activin A, probably serving a role of up-regulating activin A production locally in bone marrow microenvironment. At present, activin A is not known to play any role in inflammatory reaction; this study may thus raise the possibility that activin A performs more functions than are currently recognized. Alternatively, the enhanced production of this molecule in the bone marrow microenvironment may be regarded as a compensatory mechanism in host defenses, countering inflammatory mediators that are known to suppress erythropoiesis.

Engraftment of human T-cell acute lymphoblastic leukemia in immunodeficient NOD/SCID mice which have been preconditioned by injection of human cord blood.

Childhood T‐cell acute lymphoblastic leukemia (T‐ALL) is one of the most common childhood cancers. Study of leukemia biology, as well as preclinical testing of potential therapeutic regimens directed at T‐ALL, has been impeded by the lack of an efficient in vivo model of primary leukemia. We have reported elsewhere some observations that human cord blood conditioned medium enhances leukemia colony formation in vitro and preconditioning of sublethally irradiated nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice with cord blood mononuclear cells (MNCs) facilitates the subsequent engraftment of primary T‐ALL cells in these mice. Here we characterize in greater detail this in vivo xenograft model of human leukemia in NOD/SCID mice. Consistent with the thesis that cord blood facilitates engraftment, the engraftment of human leukemia can be shown to increase with increasing number of cord blood MNCs injected. In addition, we documented the expression of chemokine receptor CXCR4 by primary T‐ALL from patients and found that the presence of these receptors did not result in the transmigration of T‐ALL cells induced by stromal cell‐derived factor‐1α. Finally, we show that in this xenograft system T‐ALL cells recovered from engrafted bone marrow are characterized by upregulated expression of interleukin 2 receptor γ chain, suggesting that cord blood preconditioning may function in part to increase T‐ALL responsiveness to growth factor(s).

Analysis of activin A gene expression in human bone marrow stromal cells

Activin A, a member of the TGF‐β superfamily, plays roles in differentiation and development, including hematopoiesis. Our previous studies indicated that the expression of activin A by human bone marrow cells and monocytes is highly regulated by inflammatory cytokines and glucocorticoids. The present study was undertaken to investigate the regulation of activin A gene expression in the human bone marrow stromal cell lines L87/4 and HS‐5, as well as in primary stromal cells. Northern blots demonstrated that, like primary stromal cells, the cell lines expressed four activin A RNA transcripts (6.4, 4.0, 2.8, and 1.6 kb), although distribution of the RNA among the four sizes varied. The locations of the 5′ ends of the RNAs were investigated by Northern blots and RNase protection assays. The results identified a transcription start site at 212 nucleotides upstream of the translation start codon. In addition, luciferase expression assays of a series of deletion constructs were used to identify regulatory sequences upstream of the activin A gene. A 58 bp upstream sequence exhibits promoter activity. However, severalfold higher expression requires a positive element consisting of an additional 71 bp of the upstream region. Promoter activity was also identified between 2.5 and 3.6 kb upstream of the start codon. These findings suggest that expression of activin A at the transcriptional level follows complex patterns of regulation. J. Cell. Biochem. 70:8‐21, 1998.

Truncated activin type II receptor inhibits erythroid differentiation in K562 cells

Two receptor serine/threonine kinases (types I and II) have been identified as signaling transducing activin receptors. We studied the possibility of inhibiting activin A‐dependent differentiation in K562 cells, using a dominant negative mutant of type II receptor. A vector was constructed expressing activin type II truncated receptor (ActRIIa) that lacks the cytoplasmic kinase domain. Since activin type I and II receptors form heteromeric complexes for signaling, the mutant receptors compete for binding to endogenous receptors, hence acting in a dominant negative fashion. K562 cells were stably transfected with ActRIIa, and independent clones were expanded. The truncated cDNA was integrated into the genome of the transfectants, as shown by polymerase chain reaction; and the surface expression of truncated receptors was shown by affinity cross‐linking with 125I‐activin A. In wild‐type K562 cells, activin A induced erythroid differentiation and cells started to express hemoglobins. In transfected cells expressing ActRIIa, the induction of erythroid differentiation was abrogated and less than 10% of cells were hemoglobin‐containing cells after culture with activin A. Further transfection with wild‐type type II receptors rescued the mutant phenotype of these transfectants, indicating that the effect of ActRIIa is dominant negative. In addition, phosphorylation of the cytoplasmic kinase domain of the type II receptor in vitro confirms the autophosphorylation of this portion of the receptor. Therefore, induction of erythroid differentiation in vitro is mediated through the cell surface activin receptor, and interference with this receptor signaling inhibits this process of differentiation in K562 cells. J. Cell. Biochem. 78:24–33, 2000.

Human cord blood conditioned medium enhances leukemia colony formation in vitro

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. Recently, we observed that pre-conditioning sub-lethally irradiated immunodeficient mice with human cord blood mononuclear cells facilitates in these mice high level engraftment of primary T-ALL cells obtained from patients. Here we report that human cord blood cells secrete a factor(s) which markedly enhances in vitro both colony number and burst size of the T-ALL clonogenic progenitors from patients. The enhancing activity does not correspond to IL-2, IL-15, nor to several other cytokines implicated in T cell proliferation/activation. Thus, it is possible cord blood may secrete an as yet unidentified factor(s) acting on leukemia clonogenic progenitors of T-ALL. Collectively, these studies should prove invaluable in addressing the growth properties of primary T-ALL cells from patients.

Phenotypic and functional characterization of a new human macrophage cell line K1m demonstrating immunophagocytic activity and signalling through HLA class II

A human macrophage line, designated K1m, has been established from peripheral blood. K1m expresses a number of lineage‐specific markers as well as a broad array of intercellular adhesion molecules. In particular, K1m expresses high levels of human leucocyte antigen (HLA) class I and class II. In response to ligation of HLA class II (HLA‐DR), but not in response to ligation of HLA class I, K1m forms tighter homotypic aggregates and develops a striking ‘stellate’ culture phenotype. K1m also expresses Fc receptors for immunoglobulin G (IgG) (CD64, CD32, and CD16) and can be shown to phagocytose polystyrene latex beads, as well as neuroblastoma cells in the presence of tumour‐specific monoclonal antibody (mAb). The K1m cell line should therefore prove useful for studying both signalling through macrophage HLA class II and immunophagocytosis.