CCAAT/Enhancer binding protein ß controls androgen-deprivation-induced senescence in prostate cancer cells.

The processes associated with transition to castration-resistant prostate cancer (PC) growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) ß function is frequently deregulated in human malignancies and interestingly, androgen-sensitive PC cells express primarily the liver-enriched inhibitory protein isoform. We found that C/EBPß expression is negatively regulated by androgen receptor (AR) activity and that treatment of androgen-sensitive cell lines with anti-androgens increases C/EBPß mRNA and protein levels. Accordingly, we also find that C/EBPß levels are significantly elevated in primary PC samples from castration-resistant compared with therapy-naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the AR to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPß is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPß promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPß expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPß under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci and increased numbers of Ki67-positive cells. Ectopic expression of C/EBPß caused pronounced morphological changes, reduced PC cell growth and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to PC cell survival under androgen deprivation, and C/EBPß-deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen deprivation. Our data demonstrate that upregulation of C/EBPß is critical for complete maintenance of androgen deprivation-induced senescence and that targeting C/EBPß expression may synergize with anti-androgen or chemotherapy in eradicating PC.

Mechanisms of differential transferrin receptor expression in normal hematopoiesis.

We have investigated the expression of transferrin receptor (TfR) iron regulatory protein-1 (IRP-1) and iron regulatory protein-2 (IRP-2) in liquid suspension culture of purified hematopoietic progenitor cells (HPCs) induced by a growth factor stimulus to proliferation and unilineage differentiation/maturation through the erythroid, granulocytic, monocytic and megakaryocytic lineages. In initial HPC differentiation, TfR expression is induced in both erythroid and granulopoietic cultures. In late HPC differentiation (i.e. starting from day 5 of culture) and then differentiated precursor maturation, the TfR gene is highly expressed in the erythroid lineage, whereas it is sharply downmodulated in the granulopoietic, monocytopoietic and megakaryocytic series. The elevated TfR expression in erythroid cells is: (a) mediated through a high rate of TfR gene transcription; (b) modulated by intracellular iron levels; (c) mediated by TfR mRNA stabilization through the iron regulatory protein (IRP), in that IRP-1 activity is high in erythroid lineage as compared to the levels observed in other hemopoietic lineages; and (d) dependent on exogenous erythropoietin (Epo) (this is indicated by the marked TfR and IRP-1/IRP-2 downmodulation after Epo starvation). Interestingly, analysis of IRP-1 and IRP-2 expression during hemopoietic differentiation showed that: (a) IRP-1 expression was maintained during all steps of erythroid differentiation, while it was lost in the other hemopoietic lineages; (b) IRP-2 expression was observed during all stages of hemopoietic differentiation in all four lineages. However, IRP-1 and IRP-2 expression and activity are induced when monocytes, which express only low levels of IRP-1 and IRP-2, are induced to maturation to macrophages. These studies indicate that: (a) in normal erythropoiesis, the hyperexpression of TfR, starting from early erythroid HPC differentiation, is Epo-dependent and mediated via transcriptional and post-transcriptional mechanisms; (b) in the granulopoietic, monocytopoietic and megakaryocytic pathways, the TfR is first induced and then downmodulated (the latter phenomenon is mediated via transcriptional suppression of the TfR gene and IRP inactivation).

Induction of CD69 antigen on normal CD4+ and CD8+ lymphocyte subsets and its relationship with the phenotype of responding T-cells.

We evaluated phenotype and apoptotic status of normal CD4+CD69+ and CD8+CD69+ peripheral blood T-lymphocytes after short-term challenge with escalating concentrations of phytohemagglutinin (PHA). The frequency of CD69-coexpressing CD4+ and CD8+ T-cells and CD69 staining intensity increased following T-cell mitogenic stimulation; these changes were proportional to PHA concentration in culture medium. A considerable fraction of lymphocytes underwent blast transformation, displaying increased forward and side scatter signals. Interestingly enough, PHA-responsive T-cells exhibited a predominantly CD25negCD38negTCRalphabetapos phenotype; APO-1/Fas antigen (CD95) could be detected on a minority of activated CD69+ T-cells. A considerable proportion of CD69+ lymphocytes expressed intracellular perforin; in addition, an average 16+/-6% CD69+ T-lymphocytes were apoptotic after 4 h of stimulation, as evaluated by 7-amino-actinomycin-D staining and by annexin-V binding. CD69+ activated lymphocytes comprise phenotypically heterogeneous cell subpopulations potentially devoted to diverse immunological functions, i.e., proliferation, apoptosis, or cell cytotoxicity; moreover, our findings indicate that CD69 expression is proportional to the intensity of the activating stimulus and that the capacity to upregulate CD69 antigen following short-term mitogenic challenge may be restricted to unactivated CD38negCD25negTCRalphabetapos T-lymphocytes.

Expression of thrombospondin receptor (CD36) in B-cell chronic lymphocytic leukemia as an indicator of tumor cell dissemination.

BACKGROUND AND OBJECTIVE: The expression of CD36 antigen has not been conclusively associated with human B-lymphocytes although CD36 was recently detected in a human B-cell angiotropic lymphoma where it might be involved in lymphoblast-endothelial cell adhesion. We investigated the expression of CD36 in B-cell chronic lymphocytic leukemia (CLL) by multiparameter flow cytometry; results were correlated with clinical features. DESIGN AND METHODS: CD36 expression was evaluated on peripheral blood and bone marrow samples from 24 patients affected by CD5+ B-CLL. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation, were labeled with fluorochrome-conjugated monoclonal antibodies under standard experimental conditions and were analyzed by flow cytometry. CD36 expression was quantified both in terms of frequency of CD19+CD36+ cells and of mean fluorescence intensity (MFI-R) of CD36+ cell populations. The intensity of CD36 expression was arbitrarily classified as weak (MFI-R ranging from 3 to 6; score 0), moderate (MFI-R ranging from 6 to 9; score 1), intermediate (MFI-R ranging from 9 to 11; score 2) or strong (MFI-R ranging from 11 to 17; score 3). RESULTS: CD36 could be detected on 3% (range 2-5) of normal CD19+ B-lymphocytes and on 45% (range 30-75) of neoplastic CD19+ B-cells. When CLL patients were stratified according to CD36 staining intensity, higher hemoglobin levels (Hb) were recorded in patients assigned to score 0 (Hb = 14.3 g/dL; range 13.9-15.1) compared to patients scoring 1-2 (Hb = 11.2; range 10.3-12.2) or 3 (Hb = 9.8; range 9.6-11.6; p=0.0053). Similarly, higher platelet counts (Plt) were found in patients scoring 0 (Plt = 282x10(3)/microL; range 244-319), compared to patients with intermediate (Plt = 175x10(3)/microL; range 144-238) and high scores (Plt = 149x10(3)/microL; range 103-230; p=0.044); lymphocyte count (Ly) was significantly higher in patients assigned to score 3-4 (Ly = 23.3x10(3)/microL, range 13-30) compared to score 0-2 (Ly = 9.8x10(3)/microL, range 8.5-10.8; p=0.045). CLL patients expressing CD36 at intermediate-to-strong intensity (MFI-R = 14, range 9-16) were more frequently assigned to Rai stages III-IV than stages I-II (CD36 MFI-R = 9, range 6.5-11; p=0.005) and stage 0 (CD36 MFI-R = 6, range 4-7.3; p<0.001). Interestingly, bone marrow diffuse histology was strongly associated with higher CD36 expression (MFI-R = 8.7; range 4.7-13.9) compared to non-diffuse patterns of bone marrow infiltration (MFI-R = 6.7; range 5.2-9.3; p=0.0019). In multivariate regression analysis, CD36 staining intensity significantly and independently correlated with diffuse BM histology (p=0.033). INTERPRETATION AND CONCLUSIONS: The present report provides the first evidence of CD36 expression on CD19+ B-cells from CLL; the correlations with clinical parameters strongly support the view that CD36 might favor tumor cell spreading. Whether high CD36 expression levels on CLL CD19+ B-cells identify an aggressive disease subset remains to be further confirmed in larger series of patients.

The synergistic effect of simultaneous addition of retinoic acid and vitamin D3 on the in-vitro differentiation of human promyelocytic leukemia cell lines could be efficiently transposed in vivo.

Both human cell lines HL-60 and AML-193 exhibit a myeloblastic and promyelocytic morphology, respectively, but may be regarded as bipotent leukemic precursors. They can be triggered to differentiate to either granulocytes or monocytes upon retinoic acid (RA) or 1,25-dihydroxyvitamin D (D3) addition, respectively. We have investigated the effect of combined addition of these chemical inducers on the in-vitro differentiation of both cell lines. RA and D3 added together exert synergistic effects on the in-vitro maturation of these myeloid cell lines. Interestingly, the additive effects were lost if the cells were incubated with the inducers added at sequential times. The synergistic effect could be transposed in vivo and could be clinically significant in the treatment of the promyelocytic leukemia. This clinical strategy may help to prevent retinoic acid resistance or to overcome it in patients relapsed after RA therapy and usually unresponsive to a reinduction therapy with RA alone.

Terminal megakaryocytic differentiation of TF-1 cells is induced by phorbol esters and thrombopoietin and is blocked by expression of PML/RARalpha fusion protein.

We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARalpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARalpha expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in 'wild-type' TF-1 cells. Conversely, when PML/RARalpha expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARalpha gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARalpha expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.

Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors.

We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem cells (HPCs/HSCs) stringently purified from adult peripheral blood and grown in different culture systems. In these experiments HPCs/HSCs were treated with FL +/- kit ligand (KL) +/- monocyte colony-stimulatory factor (M-CSF). In clonogenetic HPC culture supplemented with interleukin-3 (IL-3)/granulomonocyte-CSF (GM-CSF)/erythropoietin (Epo), FL potentiates colony-forming unit (CFU)-GM proliferation in terms of colony number and size, but exerts little effect on burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythroid megakaryocyte macrophage (CFU-GEMM) growth, whereas KL enhances the proliferation of all HPC types; combined FL+KL +/- M-CSF treatment causes a striking shift of CFU-GM colonies from granulocytic to monocytic differentiation. In liquid suspension HPC culture, FL alone induces differentiation along the monocytic and to a minor extent the basophilic lineages, whereas M-CSF alone stimulates prevalent monocytic differentiation but little cell proliferation: combined M-CSF+FL treatment causes both proliferation and almost exclusive monocytic differentiation (97% monocytes in fetal calf serum-rich (FCS+) culture conditions, mean value). At primitive HPC level, FL potentiates the clonogenetic capacity of colony-forming units-blast (CFU-B) and high proliferative potential colony-forming cells (HPP-CFC) in primary and secondary culture; KL exerts a similar action, and additive effects are induced by FL combined with KL. More important, addition of FL alone causes a significant amplification of the number of long-term culture-initiating cells (LTC-ICs), ie, putative repopulating HSCs, whereas this effect is not induced by KL. The FL effects correlate with flt3 mRNA expression in HPCs differentiating throught the erythroid or GM pathway in liquid suspension culture: (1) flt3 mRNA is expressed in freshly purified, resting HPCs; after growth factor stimulus the message (2) is abruptly down-modulated in HPC erythroid differentiation, but (3) is sustainedly expressed through HPC GM differentiation and abolished in GM precursor maturation. This pattern contrasts with the gradual downmodulation of c-kit through both erythroid and GM HPC differentiation. The results indicate that FL exerts a stimulatory action on primitive HPCs, including a unique expanding effect on putative stem cells, whereas its distal proliferative/differentiative action is largely restricted to CFU-GM and monocytic precursors. The latter effect is potentiated by KL and M-CSF, thus suggesting that the structural similarities of FL, KL, M-CSF, and their tyrosine kinase receptors may mediate positive interactions of these growth factors son monocytic differentiation.

Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis--i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilinage differentiation/maturation through the erythroid or granulocytic lineage. In the initial HPC differentiation stages, the RB gene is gradually induced at the mRNA and protein level in both erythroid and granulopoietic cultures. In late HPC differentiation and then precursor maturation, RB gene expression is sustained in the erythroid lineage, whereas it is sharply downmodulated in the granulocytic series. Functional studies were performed by treatment of HPC differentiation culture with phosphorothioate antisense oligomer targeting Rb mRNA; coherent with the expression pattern, oligomer treatment of late HPCs causes a dose-dependent and selective inhibition of erythroid colony formation. These observations suggest that the RB gene plays an erythroid- and stage-specific functional role in normal adult hematopoiesis, particularly at the level of late erythroid HPCs.

Combined vitamin D3/retinoic acid induction of human promyelocytic cell lines: enhanced phagocytic cell maturation and hybrid granulomonocytic phenotype.

Studies on the effect of retinoic acid (RA) and 1,25-dihydroxyvitamin (D3) on the differentiation of leukemic cells have provided insight into the cellular and molecular mechanisms underlying hematopoietic cell differentiation. We have evaluated the combined effect of these chemical inducers on the differentiation of HL-60 and AML-193 promyelocytic leukemia cell lines. Simultaneous RA+D3 addition potentiated leukemic cell maturation up to mature phagocytic cells. Interestingly, AML-193 cells induced with D3 and RA displayed a typical neutrophilic morphology while exhibiting properties specific to monocytic cells, e.g., high expression of CD14 membrane antigen, capacity to bind bacterial lipopolysaccharide, and monocytic-specific esterase activity; this hybrid granulomonocytic (GM) phenotype was not observed upon initial incubation with one inducer and later addition of the other. Parallel control studies were performed with purified normal GM progenitors, triggered by interleukin 3+GM-colony-stimulating factor (CSF) in FCS-rich or -free clonogenic culture, by GM-CSF+M-CSF in FCS-rich clonogenic culture, and by M-CSF in liquid suspension culture. The progenitors grown in the first condition generate exclusively G clones, even upon addition of D3 and/or RA. The progenitors grown in the second and third culture conditions generate either G and M clones (second culture condition) or a population of cells composed by a majority of monocytes (third culture condition); the D3 addition did not modify this differentiation pattern, whereas RA or RA+D3 addition elicited a marked inhibition of monocytic differentiation. These observations suggest that the development of a hybrid GM phenotype is restricted to the progeny of bipotent GM leukemic precursors.

Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis.

We have explored the expression of the transcription factors GATA-1, GATA-2, and NF-E2 in purified early hematopoietic progenitor cells (HPCs) induced to gradual unilineage erythroid or granulocytic differentiation by growth factor stimulus. GATA-2 mRNA and protein, already expressed in quiescent HPCs, is rapidly induced as early as 3 h after growth factor stimulus, but then declines in advanced erythroid and granulocytic differentiation and maturation. NF-E2 and GATA-1 mRNAs and proteins, though not detected in quiescent HPCs, are gradually induced at 24-48 h in both erythroid and granulocytic culture. Beginning at late differentiation/early maturation stage, both transcription factors are further accumulated in the erythroid pathway, whereas they are suppressed in the granulopoietic series. Similarly, the erythropoietin receptor (EpR) is induced and sustainedly expressed during erythroid differentiation, although beginning at later times (i.e., day 5), whereas it is barely expressed in the granulopoietic pathway. In the first series of functional studies, HPCs were treated with antisense oligomers targeted to transcription factor mRNA: inhibition of GATA-2 expression caused a decreased number of both erythroid and granulocyte-monocytic clones, whereas inhibition of NF-E2 or GATA-1 expression induced a selective impairment of erythroid colony formation. In a second series of functional studies, HPCs treated with retinoic acid were induced to shift from erythroid to granulocytic differentiation (Labbaye et al. 1994. Blood. 83:651-656); this was coupled with abrogation of GATA-1, NF-E2, and EpR expression and conversely enhanced GATA-2 levels. These results indicate the expression and key role of GATA-2 in the early stages of HPC proliferation/differentiation. Conversely, NF-E2 and GATA-1 expression and function are apparently restricted to erythroid differentiation and maturation: their expression precedes that of the EpR, and their function may be in part mediated via the EpR.

Promyelocytic leukemia-specific PML-retinoic acid alpha receptor fusion protein interferes with erythroid differentiation of human erythroleukemia K562 cells.

Acute promyelocytic leukemia (APL) is characterized by a t(15;17) chromosomal translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We show here that expression of the PML-RAR alpha protein in K562 erythroleukemia cells results in a reduced expression of erythroid differentiation markers and a reduced sensitivity to the erythroid differentiative action of heme. Overexpression of RAR alpha, but not of PML, elicited a similar inhibition of K562 erythroid differentiation. These findings indicate that overexpression of either RAR alpha or PML/RAR alpha interferes with erythroid differentiation and support the hypothesis that RAR alpha is involved in the regulation of normal hematopoiesis and alteration of the RAR alpha signaling by PML/RAR alpha is implicated in the promyelocytic leukemogenesis.

PML/RAR alpha+ U937 mutant and NB4 cell lines: retinoic acid restores the monocytic differentiation response to vitamin D3.

We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced with the acute promyelocytic leukemia specific PML/RAR alpha fusion gene, the expression of which is under the control of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTPR9 cells treated with Zn2+ hence exhibit levels of PML-RAR alpha protein as high as fresh acute promyelocytic leukemia blasts. In the absence of Zn2+, i.e., upon low level PML/RAR alpha expression, 1,25-dihydroxyvitamin D3 (D3) and particularly D3 plus transforming growth factor beta 1 (TGF-beta 1) induced terminal differentiation of MTPR9 cells (as observed in "wild-type" U937 cells), on the basis of morphology, membrane antigen pattern, and functional criteria. Conversely, in the presence of Zn2+, D3 and D3 plus TGF-beta 1 failed to induce terminal differentiation, as evaluated by the above parameters. Interestingly, retinoic acid (RA) treatment suppresses the differentiation blockade induced by high level PML-RAR alpha protein; indeed, Zn(2+)-treated MTPR9 cells incubated with RA plus D3 exhibited significant terminal monocytic maturation, comparable to that of cells treated with D3 alone or combined with RA in absence of Zn2+. Similar observations were made in NB4, a PML-RAR+ human acute leukemic line. As expected RA treatment of NB4 cells causes granulocytic differentiation. Interestingly, the cell line is only scarcely induced to mature monocytic cells by D3 or D3 plus TGF-beta 1 treatment, whereas it is effectively induced to monocytic maturation by combined treatment with D3 and RA. Accordingly, the rate of NB4 cell proliferation is only slightly affected by D3 or D3 plus TGF-beta 1 treatment, mildly inhibited by RA, and markedly decreased by D3 plus RA. These results indicate that in both U937 and NB4 cells high level PML/RAR alpha expression inhibits the monocytic terminal differentiation program triggered by D3 or D3 plus TGF-beta 1, whereas RA treatment effectively antagonizes this inhibitory PML-RAR alpha action and restores the D3 differentiative effect.

Enhanced production of LPS-induced cytokines during differentiation of human monocytes to macrophages. Role of LPS receptors.

In vitro cultivated human monocytes isolated from normal peripheral blood show a time-dependent differentiation into macrophages characterized by an increased expression of transferrin receptors, CD11/CD18, and CD14 Ag. We measured the secretion of TNF-alpha and IL-6 in freshly isolated monocytes and in differentiated macrophages after LPS treatment. Differentiated macrophages produced significantly higher amounts of TNF-alpha and IL-6 than freshly isolated monocytes. This increased secretion was not a result of an enhanced accumulation of TNF-alpha and IL-6 mRNA, as comparative levels of these transcripts were found in both cell types after LPS treatment. Furthermore, LPS did not induce an antiviral state to VSV3 in monocytes, but it reduced by 3 to 5 log10 the virus yield in differentiated macrophages. The addition of antibodies to IFN-beta completely inhibited the LPS-induced antiviral state to VSV, but antibodies to IFN-alpha, TNF-alpha, or IL-6 were ineffective. A marked accumulation of IFN-beta mRNA was found in both cell types after LPS treatment. Binding experiments with FITC-LPS revealed a slightly higher overall binding affinity for LPS in freshly explanted monocytes as compared with differentiated macrophages, even though the maximal binding was higher in macrophages. In both cell types, the LPS binding was partially inhibited by antibodies to CD14. These results demonstrate that: 1) in vitro differentiation of human monocytes to macrophages leads to an enhanced LPS response in terms of (a) progressive increase of IL-6/TNF-alpha production and (b) acquisition of an IFN-beta mediated antiviral state; 2) this enhanced response to LPS, largely CD14-independent, is not linked to any increased accumulation of cytokine mRNA, but is probably a result of an increased synthesis and/or secretion of these cytokines.

Transforming growth factor-beta potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines.

We have investigated the effects of 1,25-dihydroxyvitamin D3 (D3) and/or transforming growth factor (TGF)-beta on one monocytic (U-937) and two human promyelocytic (HL-60 and AML-193) leukemic cell lines. D3 addition induces a partial monocytic maturation of the cell lines, whereas TGF-beta treatment is largely ineffective. Combined treatment with TGF-beta and D3 causes terminal monocytic maturation, as evaluated both by assessment of a large spectrum of membrane Ag and by functional assays. Furthermore, sequential addition of the two inducers showed that pretreatment with TGF-beta 1 followed by incubation with D3, but not vice versa, induces monocytic maturation as effectively as simultaneous treatment with both agents. In liquid culture the proliferative activity of these cell lines is slightly decreased by D3 and virtually unaffected by TGF-beta, whereas combined treatment with D3 and TGF-beta induces a markedly potentiated inhibitory effect. Furthermore, TGF-beta/D3 treatment (but not D3 alone) elicits the expression of membrane CD14, FcRI, FcRII, CD11a, CD11b, CD11c, ICAM-1, and PECAM-1 Ag at a level comparable to that observed on normal human monocytes. It is noteworthy that several of these Ag play an important role in monocyte physiology (e.g., CD14 Ag mediates the binding of bacterial LPS to monocytes). Treatment with both TGF-beta and D3 (but not D3 alone) induces superoxide anions and H2O2 production similar to that of circulating monocytes. In semisolid culture, D3 and TGF-beta alone cause, respectively, a marked and slight loss of cloning efficiency of the cell lines, whereas their combined addition synergistically results in a complete loss of the cloning capacity. These findings suggest a physiologic role for TGF-beta in monocyte maturation. Furthermore, they may pave the way to the design of clinical protocols combining D3 and TGF-beta in the differentiation therapy of acute promyelocytic/myelomonocytic leukemia.

c-kit ligand reactivates fetal hemoglobin synthesis in serum-free culture of stringently purified normal adult burst-forming unit-erythroid.

We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)-free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (< 10% and < 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL-3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL-induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL-3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.