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.

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 (125)I-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.

Functional and biochemical characterization of a novel human macrophage-derived negative regulator of haematopoiesis.

It is believed that haematopoiesis is regulated by both positive and negative signals derived from the marrow microenvironment, which includes macrophages. The identity and mechanism of action of the proteins mediating negative regulation is an area of active investigation. We report here the identification and initial characterization of a novel suppressor of early haematopoietic progenitors, designated NRH (for Negative Regulator of Haematopoiesis), isolated from the recently established human macrophage line 2MAC. The mechanism of NRH suppression appears to involve a marked decrease in the cycling of early progenitor cells. NRH activity was shown to be reversible and to correspond to an acidic, heparin-binding glycoprotein with a molecular weight of approximately 20 000 daltons ( approximately 20 kDa). By exploiting lectin specificity, hydrophobic interaction, and heparin affinity, we have developed a procedure for the rapid isolation of highly purified NRH from 2MAC-conditioned medium. By a number of functional and biochemical criteria, NRH appears to represent a novel macrophage-derived negative regulator of haematopoiesis which may have future application in certain clinical settings as a chemoprotectant of primitive haematopoietic cells.

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

Activin A, a member of the TGF-beta 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.

Suppression of IL-6 biological activities by activin A and implications for inflammatory arthropathies.

Activin A is a cytokine whose multiple functions have yet to be fully determined. In this study, the role of proinflammatory cytokines in regulatory control of activin A production was shown in synoviocytes and chondrocytes. Additional facets of functional inflammation-related activities of activin A were also determined. Results showed that activin A concentrations in the synovial fluid of patients with rheumatoid arthritis and gout were elevated relative to those in patients with osteoarthritis. Further studies showed that production of activin A by synoviocytes and chondrocytes in culture was stimulated by cytokines such as IL-1, transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), and IL-8, consistent with previous studies in regard to the control of activin A production in marrow stromal cells and monocytes by cytokines, glucocorticoids and retinoic acid. In addition, the relationship of activin A to IL-6-induced biological activities was investigated. Three major IL-6 activities involved in inflammatory responses were found to be suppressed by activin A. In a dose-dependent manner, activin A efficiently suppressed IL-6-induced proliferation of 7TD1 B lymphoid cells, phagocytic activity of monocytic M1 cells, and fibrinogen production in HepG2. Therefore, it is likely that activin A serves as a suppressor for IL-6, dampening inflammatory responses, and has the potential to perform some previously unrecognized roles in inflammation.

Contrasting effects of inflammatory cytokines and glucocorticoids on the production of activin A in human marrow stromal cells and their implications.

Human marrow stromal cells were analysed with immunocytochemical staining, Northern blot, and functional bioassay for production of activin A. Although Northern blot and immunocytochemical staining did not detect the alpha subunit of inhibin in human marrow stromal cells, RT-PCR analyses confirmed its presence, along with the expected activin beta A PCR products. Present studies showed that human marrow fibroblastoid cells were reactive with anti-activin A antibodies and that the production of beta A RNA was upregulated by pro-inflammatory cytokines/regulators like interleukin 1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) or 12-O-tetradecanoylphorbol 13-acetate (TPA). IL-1 alpha or TNF-alpha stimulated-marrow stromal cells accumulated beta A RNA after 2 h of incubation, reaching a peak stimulation at approximately 8 h. Biologically active activin A molecules were detected in the conditioned media by a bioassay, and their activity was specifically inhibited by a blocking antibody or an activin-binding protein, follistatin. Accumulation of bioactive activin A in conditioned medium of human marrow stromal cells increased after incubation with IL-1 alpha or TNF-alpha. Nuclear run-off assays with TNF-alpha stimulated marrow stromal cells showed that the enhanced expression of activin A was related to an increase in its rate of transcription. In contrast to the stimulatory effect of pro-inflammatory cytokines, hydrocortisone and dexamethasone at 1 x 10(-7) to 1 x 10(-6) M inhibited both the constitutive and the cytokine-stimulated expression of activin beta A RNA, and also the production of bioactive activin A protein. The upregulation of activin A production by cytokines and its suppression by glucocorticoids imply that activin A may also act as a moderator in diverse functions including host defences.

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 1, 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.

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 beta A beta A in these monocytes was further confirmed by 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 x 10(-7) to 1 x 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.

Clinical significance of p53 mutations in relapsed T-cell acute lymphoblastic leukemia.

In T-cell acute lymphoblastic leukemia (T-ALL), p53 gene mutations were found in 12 of 51 patients in first relapse (24%). In a retrospective study, bone marrow samples at diagnosis were obtained from 9 of the 12 relapsed patients with p53 mutation; only one patient was found to harbor a p53 mutation at diagnosis. No further p53 mutations were identified in 18 unpaired diagnosis T-ALL samples. This is the first report of a p53 mutation in T-ALL at diagnosis. p53 mutations in relapsed T-ALL were clinically relevant. Patients with p53 mutations experience a shorter duration of survival than those patients without p53 mutations. Additionally, patients with p53 mutations were significantly less likely to have achieved a complete second remission from reinduction therapy than those patients without p53 mutations and experience a shorter duration of survival from relapse even when a second reinduction is obtained. Though primarily identified only at relapse, p53 mutations were also associated with a decreased duration of first remission and overall decrease in survival from diagnosis. Patients with p53 mutations had a 3.8-fold increase in risk of death than those patients without p53 mutations. These findings suggest that p53 mutation is associated with poor clinical outcome that is characterized by (1) a shortened duration of survival after first relapse; (2) a reduced response to reinduction therapy; (3) a shortened duration of first remission; and, hence, (4) an overall decreased duration of survival and increased risk of death.

Detection of functional and dimeric activin A in human marrow microenvironment. Implications for the modulation of erythropoiesis.

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.

Sensitivity of committed hematopoietic progenitor cells in vitro (BFU-E, CFU-E, CFU-GM) and two human carcinoma cell lines toward rhodamine-123 and phosphonium salt II-41.

Lipophilic cationic compounds accumulate more rapidly in the mitochondria of many carcinoma-derived cells than in non-transformed cells, thus leading to their pronounced cytotoxic effects on carcinoma cells. In this report, in order to measure tumoricidal effects vs cytotoxicity to normal hematopoietic progenitors, we studied the sensitivity of committed human hematopoietic cells in vitro and two human carcinoma cell lines (2008 ovary carcinoma cells and HT29 colon cells) toward two such compounds, rhodamine-123 and phosphonium salt II-41. Continuous exposure of human marrow cells to rhodamine-123 or phosphonium salt II-41 for 7 and 14 days produced dose-related inhibition of colony formation of erythroid burst-forming units (BFU-E), erythroid colony forming units (CFU-E), and CFU-granulocyte/macrophage (CFU-GM). The average values of IC50 for several different human bone marrows are approximately 0.9-1.1 microM for rhodamine-123 toward BFU-E, CFU-E and CFU-GM, and 31-38 microM for phosphonium salt II-41 toward the same hematopoietic progenitors. These IC50 values are similar for each type of hematopoietic progenitors. In each case, rhodamine-123 appears to be at least 30-fold more growth suppressive than phosphonium salt II-41 in these in vitro colony assays. In addition, the sensitivity of these hematopoietic progenitors toward these two compounds is comparable to the inhibition of colony formation for the two human carcinoma cell lines. The lack of differences in the sensitivity among the various hematopoietic progenitors in vitro may disagree with previous studies showing there are vast differences in the state of cell cycle for these hematopoietic progenitor cells. However, these observations about the cytotoxicity in vitro can be explained by assuming that the cytotoxicity of these compounds depends on other factors such as differentiation processes, which result in the appearance of many or very active mitochondria. Alternatively, the lack of differences in the sensitivity of the in vitro colony formation can also be attributed to a reported decrease in expression of P-glycoprotein, a multidrug efflux pump, in the differentiating hematopoietic progeny cells.

Activin A-induced differentiation in K562 cells is associated with a transient hypophosphorylation of RB protein and the concomitant block of cell cycle at G1 phase.

The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF-beta 1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K562 cells. Activin A-treated K562 cells were found to undergo a transient block in cell cycle, temporarily halting progression from G1 to S phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell cycle, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9-24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle-related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells.

Importance of FSH-releasing protein and inhibin in erythrodifferentiation.

Inhibin is a hypophysiotropic hormone which selectively suppresses the secretion of pituitary follicle-stimulating hormone. It has been isolated from gonadal fluids and characterized as a protein heterodimer consisting of an alpha subunit and one of two beta subunits (beta A or beta B). FSH-releasing protein (FRP), also named activin, is a dimer consisting of two inhibin beta-chains. A factor from conditioned medium of a leukaemia cell line has been isolated which can induce mouse Friend cells to become benzidine-positive, and which shares a similar N-terminal sequence with porcine FRP. In this report, we find that FRP and inhibin modulate both the induction of haemoglobin accumulation in a human erythroleukaemic cell line, K562, and the proliferation of erythroid progenitor cells in human bone marrow culture. These two proteins could constitute a novel humoral regulatory control of erythropoiesis which would involve two types of related protein dimers with functionally opposite effects.