Transferrin receptor mediates uptake and presentation of hepatitis B envelope antigen by T lymphocytes.

Human activated T lymphocytes expressing class II molecules are able to present only complex antigens that bind to their own surface receptors, and thus can be captured, internalized, and processed through the class II major histocompatibility complex processing pathway. We have used the antigen-presenting T cell system to identify the viral receptor used by hepatitis B virus (HBV) to enter cells, as well as the sequence of HB envelope antigen (HBenvAg) involved in this interaction. Results show that both CD4+ and CD8+ T clones can process and present HBenvAg to class II-restricted cytotoxic T lymphocytes and that the CD71 transferrin receptor (TfR) is involved in efficient HBenvAg uptake by T cells. Moreover, we provide evidence that the HBenvAg sequence interacting with the T cell surface is contained within the pre-S2 region. Since TfR is also expressed on hepatocytes, it might represent a portal of cellular entry for HBV infection. This system of antigen presentation by T cells may serve as a model to study both lymphocyte receptors used by lymphocytotropic viruses and viral proteins critical to bind them.

The interplay between the master transcription factor PU.1 and miR-424 regulates human monocyte/macrophage differentiation.

We describe a pathway by which the master transcription factor PU.1 regulates human monocyte/macrophage differentiation. This includes miR-424 and the transcriptional factor NFI-A. We show that PU.1 and these two components are interlinked in a finely tuned temporal and regulatory circuitry: PU.1 activates the transcription of miR-424, and this up-regulation is involved in stimulating monocyte differentiation through miR-424-dependent translational repression of NFI-A. In turn, the decrease in NFI-A levels is important for the activation of differentiation-specific genes such as M-CSFr. In line with these data, both RNAi against NFI-A and ectopic expression of miR-424 in precursor cells enhance monocytic differentiation, whereas the ectopic expression of NFI-A has an opposite effect. The interplay among these three components was demonstrated in myeloid cell lines as well as in human CD34+ differentiation. These data point to the important role of miR-424 and NFI-A in controlling the monocyte/macrophage differentiation program.

Identification and expansion of the tumorigenic lung cancer stem cell population.

Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133(+) cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 10(4) lung cancer CD133(+) cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133(+) cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133(+) cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.

Biogenesis of erythropoietin: evidence for pro-erythropoietin in a subcellular fraction of kidney.

The light mitochondrial fraction of hypoxic rodent kidneys, called the renal erythropoietic factor, contains erythropoietin in a pro, or active form. Erythropoietin is released from this inactive form when the renal erythropoietic factor is incubated with normal serum. The biogenesis of erythropoietin possibly involves a system in kidney reminiscent of the proinsulin-insulin system in pancreas.

Lineage-specific requirement of c-abl function in normal hematopoiesis.

Structural abnormalities of the c-abl proto-oncogene are found in hematopoietic cells of more than 90 percent of individuals with chronic myelogenous leukemia. Therefore c-abl may be important in normal as well as malignant hematopoiesis. Normal human hematopoietic progenitor cells were exposed to three different c-abl sense or antisense oligodeoxynucleotides, and the effects on myeloid and erythroid colony formation were examined. The c-abl antisense oligodeoxynucleotides inhibited myeloid, but not erythroid, colony formation. The c-abl sense oligodeoxynucleotides and bcr sense and antisense oligodeoxynucleotides were not inhibitory in this assay. These data show that c-abl is critical in normal myelopoiesis and may explain the relatively selective expansion of leukocytes in patients with chronic myelogenous leukemia.

"Pure" human hematopoietic progenitors: permissive action of basic fibroblast growth factor.

Methodology has been developed that enables virtually complete purification and abundant recovery of early hematopoietic progenitors from normal human adult peripheral blood. A fraction of the pure progenitors is multipotent (generates mixed colonies) and exhibits self-renewal capacity (gives rise to blast cell colonies). This methodology provides a fundamental tool for basic and clinical studies on hematopoiesis. Optimal in vitro cloning of virtually pure progenitors requires not only the stimulatory effect of interleukin-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin, but also the permissive action of basic fibroblast growth factor. These findings suggest a regulatory role for this growth factor in early hematopoiesis.

KDR receptor: a key marker defining hematopoietic stem cells.

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.

A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.

PLZF-mediated control on VLA-4 expression in normal and leukemic myeloid cells.

The promyelocytic leukemia zinc-finger protein (PLZF) is a transcriptional repressor. To investigate the role of PLZF in the regulation of cytoadhesion molecules involved in the mobilization of hemopoietic cells, we have analysed PLZF and very late antigen 4 (VLA-4) expression in normal and leukemic cells. In hematopoiesis, we found a negative correlation between PLZF and VLA-4 expression, except for the megakaryocytic lineage. In contrast, we observed a positive correlation between PLZF and VLA-4 expression in a panel of acute myeloid leukemia (AML) samples. In K562 cells expressing PLZF (K562-PLZF), we found that the expression of VLA-4 and c-kit was downmodulated. We have investigated the possibility for VLA-4 or the c-kit receptor to be direct target genes of PLZF in K562-PLZF cells and identified a PLZF DNA-binding site within the VLA-4 promoter. Furthermore, decrease in VLA-4 expression was associated with loss of adhesion on fibronectin-coated plates, which promotes drug-induced apoptosis of K562-PLZF cells. Our findings indicate that VLA-4 is a potential target gene of PLZF. However, in primary AMLs the control of PLZF on VLA-4 expression is lost. Altogether, we suggest that VLA-4 modulation by PLZF may represent an important step in the control of normal and leukemic cell mobilization.

Unilineage monocytopoiesis in hematopoietic progenitor culture: switching cytokine treatment at all Mo developmental stages induces differentiation into dendritic cells.

We have developed a new culture system whereby human hematopoietic progenitors purified from adult peripheral blood extensively proliferate and gradually differentiate into >95% pure monocytic (Mo) cells. At all developmental stages treatment with interleukin (IL)-4+granulocyte-macrophage colony-stimulating factor or IL-4+c-Kit-ligand+FLT-3 ligand switched the Mo precursors into dendritic cells (DCs). The switching capacity decreased only at the end of the culture, when most Mo cells matured to macrophages. Moreover, the Mo precursors were highly susceptible to transduction with lentiviral vectors: once switched to DCs, they maintained the transgene expression, as well as the phenotype and function of the DC lineage. Our results provide new insight into the potential role of the Mo lineage as a reservoir of DCs in vivo. Furthermore, the methodology for transduction of Mo precursors provides a tool to generate genetically modified, normally functioning DCs potentially useful for immunotherapy.

Overexpression of Ets-1 in human hematopoietic progenitor cells blocks erythroid and promotes megakaryocytic differentiation.

Ets-1 is a widely expressed transcription factor implicated in development, tumorigenesis and hematopoiesis. We analyzed Ets-1 gene expression during human erythroid and megakaryocytic (MK) differentiation in unilineage cultures of CD34+ progenitor cells. During erythroid maturation, Ets-1 is downmodulated and exported from the nucleus into the cytoplasm through an active mechanism mediated by a leucine-rich nuclear export signal. In contrast, during megakaryocytopoiesis Ets-1 increases and remains localized in the nucleus up to terminal maturation. Overexpression of Ets-1 in erythroid cells blocks maturation at the polychromatophilic stage, increases GATA-2 and decreases both GATA-1 and erythropoietin receptor expression. Conversely, Ets-1 overexpressing megakaryocytes are characterized by enhanced differentiation and maturation, coupled with upmodulation of GATA-2 and megakaryocyte-specific genes. We show that Ets-1 binds to and activates the GATA-2 promoter, in vitro and in vivo, indicating that one of the pathways through which Ets-1 blocks erythroid and promotes MK differentiation is via upmodulation of GATA-2 expression.

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line.

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis.

To determine if MYB protein is preferentially required during specific stages of normal human hematopoiesis we incubated normal marrow mononuclear cells (MNC) with c-myb antisense oligodeoxynucleotides. Treated cells were cultured in semisolid medium under conditions designed to favor the growth of specific progenitor cell types. Compared with untreated controls, granulocyte-macrophage (GM) CFU-derived colonies decreased 77% when driven by recombinant human (rH) IL-3, and 85% when stimulated by rH GM colony-stimulating factor (CSF); erythroid burst-forming unit (BFU-E)- and CFU-E-derived colonies decreased 48 and 78%, respectively. In contrast, numbers of G-CSF-stimulated granulocyte colonies derived from antisense treated MNC were unchanged from controls, though the numbers of cells composing these colonies decreased approximately 90%. Similar results were obtained when MY10+ cells were exposed to c-myb antisense oligomers. When compared with untreated controls, numbers of CFU-GM and BFU-E colonies derived from MY10+ cells were unchanged, but the numbers of cells composing these colonies were reduced approximately 75 and greater than 90%, respectively, in comparison with controls. c-myc sense and antisense oligomers were without significant effect in these assays. Using the reverse transcription-polymerase chain reaction, c-myb mRNA was detected in developing hematopoietic cells on days 0-8. At day 14 c-myb expression was no longer detectable using this technique. These results suggest that c-myb is required for proliferation of intermediate-late myeloid and erythroid progenitors, but is less important for lineage commitment and early progenitor cell amplification.

Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.

Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma).

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.

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts.

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.

Proliferative response and oncogene expression induced by epidermal growth factor in EL2 rat fibroblasts.

Extensive evidence supports a two-step model for the control of fibroblast growth, which includes first the action of a competence factor (e.g., platelet-derived growth factor) followed by the stimulus of a progression factor (e.g., epidermal growth factor [EGF]). We investigated whether this model may be applied to the euploid EL2 fibroblast line recently isolated from rat embryos (E. Liboi, M. Caruso, and C. Basilico, Mol. Cell. Biol. 4:2925-2928, 1984). Our results clearly show that EGF alone leads EL2 cells to proliferate in serum-free conditions at a rate corresponding to 50 to 60% of that observed in the presence of 10% calf serum. It is of interest that, when resting EL2 cells were exposed to EGF, transcription of both c-myc and c-fos was markedly induced. Altogether, these observations suggest that, in contrast with the model of fibroblast growth mentioned above, EL2 cells require the presence of a single growth factor (EGF) for induction of DNA synthesis, and the expression of myc and fos proto-oncogenes may represent an obligatory step in the pathway of commitment of EL2 cells to proliferation. In addition, we showed that EGF may induce EL2 cells to acquire some properties of transformed cells, such as growth in agar and loss of contact inhibition. This suggests that the particular response to EGF of the EL2 line may be strictly connected with the expression of a transformed phenotype.

Overexpression of the zinc finger protein MZF1 inhibits hematopoietic development from embryonic stem cells: correlation with negative regulation of CD34 and c-myb promoter activity.

Zinc finger genes encode proteins that act as transcription factors. The myeloid zinc finger 1 (MZF1) gene encodes a zinc finger protein with two DNA-binding domains that recognize two distinct consensus sequences, is preferentially expressed in hematopoietic cells, and may be involved in the transcriptional regulation of hematopoiesis-specific genes. Reverse transcription-PCR analysis of human peripheral blood CD34+ cells cultured under lineage-restricted conditions demonstrated MZF1 expression during both myeloid and erythroid differentiation. Sequence analysis of the 5'-flanking region of the CD34 and c-myb genes, which are a marker of and a transcriptional factor required for hematopoietic proliferation and differentiation, respectively, revealed closely spaced MZF1 consensus binding sites found by electrophoretic mobility shift assays to interact with recombinant MZF1 protein. Transient or constitutive MZF1 expression in different cell types resulted in specific inhibition of chloramphenicol acetyltransferase activity driven by the CD34 or c-myb 5'-flanking region. To determine whether transcriptional modulation by MZF1 activity plays a role in hematopoietic differentiation, constructs containing the MZF1 cDNA under the control of different promoters were transfected into murine embryonic stem cells which, under defined in vitro culture conditions, generate colonies of multiple hematopoietic lineages. Constitutive MZF1 expression interfered with the ability of embryonic stem cells to undergo hematopoietic commitment and erythromyeloid colony formation and prevented the induced expression of CD34 and c-myb mRNAs during differentiation of these cells. These data indicate that MZF1 plays a critical role in hematopoiesis by modulating the expression of genes involved in this process.

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.

A pentamer transcriptional complex including tal-1 and retinoblastoma protein downmodulates c-kit expression in normal erythroblasts.

Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb(-) SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1-E12-Lmo2 and tal-1-E12-Lmo2-Ldb1 complexes but not that of a tal-1-E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1-E12-Lmo2-Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.