|
|
|
|
|
|
|
||
Articles |
,,¶,,¶ Department of Pharmacology, the Department of Biochemistry, the || Department of Microbiology-Immunology, and ¶ The Program of Molecular Biology, University of Montreal, Montreal, Quebec H3C 3J7, Canada; ** The National Cancer Institute, Naval Medical Center, Bethesda, Maryland 20892; The Department of Medicine, McGill University, Montreal, Quebec H3A 2A7, Canada; and Institut National de la Santé et de la Recherche Médicale U.91, Hôpital Henri Mondor, 94010 Créteil, France
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key Words: SCL • TAL1 • c-kit • apoptosis • Steel factor
Abbreviations used: as-SCL, antisense SCL; dn-SCL, dominant negative SCL; GAPDH, rat glyceraldehyde-3-phosphate dehydrogenase; MSCV, murine stem cell virus; SF, Steel factor.
CL, also known as TAL1 or Tcl-5, is associated with 25% of chromosomal rearrangements in childhood T cell acute lymphoid leukemia (1–6). SCL codes for a hemopoietic-specific transcription factor of the basic helix-loop-helix family, and can enhance or suppress transcription (7). SCL expression is detected in primitive pluripotent hemopoietic precursors (8, 9), in a subset of CD34+ cells, in mast cells and megakaryocytes (8, 10, 11) and in maturing erythroid cells (9, 12–16). Consistent with a role for SCL in establishing the hemopoietic lineage, a targeted disruption of the SCL gene specifically abrogates blood cell formation, resulting in embryonic lethality at day E8.5 (17, 18). Moreover, scl–/– embryonic stem cells fail to contribute to hemopoietic stem cells and blood cells in chimeric mice, further suggesting a critical role of SCL for the development of all hemopoietic lineages (19, 20). Together, these results suggest that SCL is required for mesodermal cell commitment into hemopoietic stem cells and/or is essential for the survival of hemopoietic stem cells.
SCL binds DNA only upon interaction with the ubiquitous basic helix-loop-helix transcription factors E2A or HEB (7, 21). The SCL-E2A heterodimer preferentially binds the TAL1 consensus AACAGATGGT defined by in vitro binding site selection (CASTing). SCL represses the E2A-dependent activity directed from the TAL1 consensus in front of a minimal promoter, but relieves the inhibition conferred by Id1 on E2A (7). However, the TAL1 consensus has not been found in any known hemopoietic promoter. SCL also associates with Lmo-2 or RBTN-2, a RING finger protein (22–25) in a multiprotein complex that includes E2A, GATA-1, an erythroid zinc finger transcription factor (26), and a novel LIM-binding protein, Ldb1 (24), and selectively binds an Ebox-GATA motif also defined by cycle amplification and selection of targets (CASTing). Although similar motives are found on two erythroid promoters, those of the glycophorin A and B genes and the porphobilinogen deaminase gene, their contribution to promoter activity in response to SCL has not been established (24). Finally, in T-ALL, SCL upregulates the expression of a cell surface marker, TALLA-1 (27). However, it is not clear whether TALLA-1 is a direct target of SCL. Thus, despite observations that indicate a crucial role for SCL in normal hemopoiesis and T cell leukemogenesis, no natural binding site or SCL target gene has been as yet reported.
The survival of hemopoietic cells both in culture and in vivo is critically dependent on the presence of hemopoietic growth factors that act to suppress apoptosis (28–30). The importance of such growth factors for blood cell development was first revealed by the genetics of hereditary anemias in mice. Perhaps the best studied examples are white spotting and Steel mice that are severely anemic due to mutations in the genes encoding the tyrosine kinase receptor c-kit (31) or its ligand Steel factor (SF),1 respectively (32, 33). Severe mutations result in lethality in homozygotes (for review see references 34, 35), indicating a crucial role for these two genes in hemopoiesis. We and others have previously shown that SF suppresses apoptosis in early hemopoietic cells that express the surface antigen CD34 (32, 36), and in the CD34+ cell line TF-1 (28). SF has also been shown to synergize with IL-7 in delaying apoptosis in primitive thymocytes (37–39).
The expression pattern of SCL in primary hemopoietic cells parallels that of c-kit (8), suggesting that SCL functions in concert with c-Kit. Furthermore, a two- to fourfold augmentation of SCL protein was observed in maturing erythroid progenitors stimulated with SF (16). However, it is not known whether upregulation of SCL is a cause or consequence of c-Kit activation, cell survival, or cell proliferation. Because of the correlation between c-Kit and SCL expression, we directly investigated the relationship of SCL function relative to c-Kit through the attenuation of SCL protein levels in TF-1 cells using an antisense SCL (as-SCL) expression vector, or through expression of a dominant negative SCL (dn-SCL). We reasoned that SCL function is likely to be determined by the nature of its binding partners, which have yet to be defined in CD34+ cells. We therefore chose to investigate this through the disruption of endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which should express appropriate SCL binding partners. Moreover, TF-1 has retained one of the most important characteristics of primary hemopoietic cells (9, 40), i.e., their requirement in hemopoietic growth factors for cell survival (28, 41). Our approach to defining SCL function and SCL target(s) was to disrupt SCL in TF-1 cells and to screen stable transfectants with a functional assay, i.e., cell survival in response to different growth factors.
Purified recombinant human GM-CSF was a gift from Dr. Steve Clark (Genetics Institute, Cambridge, MA) and purified recombinant SF was from Dr. K. Langley (Amgen, Thousand Oaks, CA). The monoclonal antibody 2TL136 specific for human SCL was provided by Dr. Danièle Mathieu-Mahul (INSERM, Marseille, France; references 10, 42). The monoclonal antibody ACK2 is specific for murine c-Kit (GIBCO BRL). The monoclonal mouse anti–human c-Kit was from Cedarlane Labs. (Hornby, Ontario, Canada). The monoclonal mouse anti–rabbit eIF-4E cross-reacts with the human protein (Transduction Labs., Lexington, KY). The monoclonal anti-CD45 (clone 4B2; ATCC number HB 196, American Type Culture Collection, Rockville, Maryland) was used at 1:10 dilution of hybridoma supernatant. Monoclonal anti–hGM-CSF receptor β chain was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and used at a 1:100 dilution. Annexin V was purchased from Biodesign (Kennebunk, ME), and used at a concentration of 400 mg/ml.
Retrovirus Production and Infection.
For retroviral infection, 106 exponentially growing cells (TF-1 or A31) were presensitized with polybrene at 2 µg/ml for 24 h and cocultured with virus-producing clones for another 24 h. Nonadherent TF-1 cells were separated from the infected fibroblasts. A polyclonal population was analyzed 7 d after selection in G418 at 1 mg/ml. For long-term experiments, cloning was performed through limiting dilution (12).
Plasmids and Transfection.
Stable TF-1 transfectants harboring the as-SCL were obtained through Lipofectin-mediated DNA transfer (GIBCL BRL) as previously described (9, 12). Cells were cloned immediately after gene transfer by limiting dilution, and the selective pressure was applied the following day, at a concentration of 1 mg/ml G418 (GIBCO BRL). GM-CSF was present throughout the gene transfer procedure. After selection, G418 concentrations were lowered to 400 µg/ml in order to expand the cells and prepare a large stock of frozen cells immediately after characterization. Each cell line was kept for not more than 2 mo in culture.
Nuclear Staining for SCL.
Surface Marker Staining.
Northern Blotting Analysis.
Western Blotting Analysis.
Membranes were blocked in 5% nonfat dry milk and 1% BSA, incubated with 2TL13G hybridoma tissue culture medium (monoclonal anti-SCL) diluted 1:300 for 2.5 h at room temperature, washed, incubated with a goat anti–mouse alkaline phosphatase– linked antibody (Bio-Rad), diluted 1:1,000 for 1.5 h, and washed extensively. Western blots were then developed by incubating membranes in the dark with 10 ml of BCIP (165 µg/ml) and NBT (330 µg/ml) (GIBCO BRL) for 2 min, and the reaction was terminated by several washes with water as described previously (42). In parallel, Western blotting was performed with a monoclonal antibody against the eukaryotic translation initiation factor (eIF-4E) (Transduction Labs.) as a control for protein loading.
Electrophoretic Mobility Shift Assays
c-kit Promoter Constructs, Transfection Protocols, and Luciferase Assay.
Apoptosis Assays.
Double Fluorochrome Staining Assay.
We first determined the efficiency of as-SCL in disrupting SCL protein levels, which were assessed through immunostaining of permeabilized cells with the monoclonal anti-TAL1 antibody BTL73 (Fig. 1). As expected, TF-1 cells express high levels of endogenous SCL. In contrast, 1 wk after transduction of as-SCL the endogenous SCL protein level was significantly decreased, with little if any overlap between the two populations. The cells were further analyzed for expression of several cell surface markers. Data shown in Fig. 1 indicate that the fluorescence intensity for c-Kit (CD117) was at least three- to fourfold lower in dn-SCL and as-SCL transfectants when compared with parental TF-1 cells (data not shown) or control cells expressing the vector alone (MSCV ). In contrast, the level of CD45 was not affected by as-SCL or dn-SCL expression and that of CD116 (GM-CSF receptor
Materials and Methods
Top
Abstract
Materials and Methods
Results
Discussion
References
Cell Lines, Growth Factors, and Antibodies.
The TF-1 cell line (40) was a gift from Dr. T. Kitamura (DNAX, Palo Alto, CA). The cells were maintained in IMDM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS (GIBCO BRL) and 5 ng/ml human GM-CSF. The cells were passaged every second day at 1.5 x 105/ml. Jurkat T cells were also maintained in IMDM supplemented with FCS (10%), and were passaged three times weekly at a concentration of 1.5 x 105/ml.
The human SCL cDNA, the dn-SCL devoid of DNA binding domain (12) and the as-SCL were all cloned in the EcoRI site of the murine stem cell virus (MSCV)-neo vector (43). High titer amphotropic viruses (106 PFU/ml) were produced by transient transfection into BING cells (44). The cells were irradiated and used for coculture with TF-1 cells for 48 h. Murine c-kit cloned in the LXSN retroviral vector was a gift from Dr. A. Bernstein (Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada). Ecotropic viruses were produced by transfection into the BOSC23 packaging cell line (44).
The human SCL cDNA was cloned in the antisense orientation under the control of the metallothionein promoter by digesting the plasmid pMSCL (12) with EcoRI and religation. The EcoRI sites come from the plasmid polylinker.
3 x 105 TF-1 and as-SCL transfectants were fixed in 500 µl of Bouin's fixative for 15 min at room temperature. The cells were then pelleted at 200 g for 5 min and resuspended in 200 µl of 0.2% Triton X-100, followed immediately with 800 µl of PBS. The cells were pelleted again, washed with 1 ml of PBS, and resuspended in 100 µl of 1:10 dilution of anti-SCL monoclonal antibody (clone BTL73, provided by Dr. Danièle Mathieu-Mahul). After 30 min of incubation on ice, cells were washed twice with 1 ml of PBS followed by a 10-min wash in 1 ml of PBS, resuspended in 100 µl of FITC-coupled goat anti–mouse antibody at the recommended dilution (Caltech, San Francisco, CA), and incubated for 30 min at 4°C. The cells were washed as above before flow cytometry analysis.
For surface marker staining, 3–5 x 105 cells were washed once with PBS supplemented with 2% FBS and 0.05% sodium azide (staining buffer), labeled with the primary antibody for 30 min on ice in a total volume of 100 ml, washed three times with 1 ml of staining buffer, and labeled with a biotinylated goat anti–mouse antibody (Cedarlane, Labs.) in staining buffer supplemented with 1% normal goat serum (Sigma-Aldrich, Oakville, Ontario, Canada) for 30 min on ice. After washing, the cells were incubated with PE-conjugated streptavidin (Sigma) for an additional 30 min on ice, washed, and analyzed on the FACScan® flow cytometer (Becton Dickinson, San Jose, CA).
Cells were homogenized in the presence of guanidium and RNA isolated by acid-phenol extraction as previously described (9, 45). Northern blotting and hybridization were performed as previously described (9). Probes used were the 1.2-kb Hind–XbaI human SCL cDNA fragment (nucleotides 109–1293; reference 12), the 1.4-kb SalI–XbaI fragment of human GATA-1 cDNA (26), a 1.5- kb DraI–KpnI human c-kit cDNA fragment from human c-kit–BS plasmid (gift from Dr. J. Simms, Immunex, Seattle, WA), and the 1.2-kb PstI– XbaI fragment of the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (46). Blots were exposed to a PhosphorImager screen for quantitation.
Cell lysates were prepared as described previously (42). Protein concentrations were determined with the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). Equal amounts of proteins (20 µg) were loaded on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose using the mini "Transblot" (Bio-Rad) for 1 h at 100 V.
Nuclear extracts were prepared as previously described (47). Protein concentrations were assayed with the Bio-Rad Protein Assay reagent. Binding reactions for electrophoretic mobility shift assays were performed as previously described (42). In brief, the binding reactions were allowed to proceed at room temperature for 15 min in the presence of 0.5 µg of poly(dI-dC) as nonspecific competitor DNA in 20 mM Hepes (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 10 µg BSA, 100,000 cpm double-stranded synthetic oligonucleotide, and 25 µg nuclear extract in a total volume of 10 µl. 50-fold molar excess of unlabeled oligonucleotides were used for self-competition and added before nuclear extracts. For antibody supershift assays, 200 ng of affinity-purified anti-E2A (Santa Cruz Biotechnology, Santa Cruz, CA), 1 µl of the monoclonal anti-SCL antibody (BTL73; reference 48), or 1 µl of an unrelated antibody were added to the binding reaction. Protein complexes were resolved by electrophoresis on 4% polyacrylamide gel buffered in 0.25x Tris-borate– EDTA, 195 mM glycine (pH 8.5) at 4°C. The sequences of the probes (coding strand) are: ACCTGAA CAGATG GTCGGCT TAL1 consensus (21); CTAGGGAG CACCTG CCAGGTG GCTGGCCC murine c-kit probe (49); and an E box probe derived from the rat POMC E box also referred to as DE2c (50).
The human c-kit promoter was cloned by PCR from genomic DNA to generate a fragment that extends from 1146 bp upstream of the transcription initiation site to 43 bp downstream. In brief, genomic DNA was amplified in two steps, the first PCR providing a fragment that extends from position –634 to +60 (PCR1), and the second PCR a fragment that covers –1146 to –379 (PCR2). PCR1 was cut with SalI and BamHI and PCR2 with HindIII (site added) and SalI, and the fragments were cloned into the HindIII and BglII sites of the promoterless luciferase expression vector pXPII to generate kit 1146. The chimeric construct was verified through sequencing. TF-1 and A31 cell lines were transfected by electroporation. Cells were passaged 24 h before gene transfer at a concentration of 3 x 105 cells/ml. Exponentially growing cells were then concentrated at 2.5 x 107 cells/ml and electroporated at 900 µF and 350 mV using a Bio-Rad electroporator with 12 µg of reporter DNA and 1.5 µg of CMV β-galactosidase used as an internal control for the experiment, with or without different molar ratios of MSCV-SCL as shown; the total amount of transfected DNA was then filled to 25 µg with pGEM4 as a carrier. Cell lysates were prepared 15 h after transfection, normalized for β-galactosidase content, and assayed for luciferase activity. Rous Sarcoma virus–driven luciferase (RSV-luc) was used as an external control for all the transfections and pXP2 as a negative control as described previously (47).
Analysis of DNA fragmentation by agarose gel electrophoresis was performed as previously described (28). In brief, equal cell numbers were lysed in Sarkosyl buffer and treated with Proteinase K and RNase A before loading on a 1.2% agarose gel (20 cm), followed by a 16-h electrophoresis at 40 V. Cell viability was independently confirmed through trypan blue exclusion and counting (28, 29), or the double fluorochrome staining assay, as described below. Apoptosis was also assessed by flow cytometry analysis of cells labeled with Annexin V-FITC (1 µg/ml), shown previously to be an early marker of apoptosis (51, 52). Immediately before acquisition, 1 ml of propidium iodide solution (1 mg/ml) was added in order to detect dead cells.
A stock solution of dye mix containing 100 µg/ml acridine orange and 100 µg/ml ethidium bromide was prepared in PBS. Cell were resuspended at 5 x 105 to 5 x 106 in IMDM. 1 µl of dye mix was added to 25 µl of cell suspension, and 10 µl of the mixture was placed on a microscope slide for examination by fluorescent microscopy (Leica, Wetzlar, Germany). Cells with bright green chromatin were considered viable cells while those with bright orange chromatin or collapsed chromatin were considered dead cells. A minimum of 200 cells were counted.
Results
Top
Abstract
Materials and Methods
Results
Discussion
References
Decreased SCL Protein Levels and c-Kit Levels in TF-1 Cells Expressing an as-SCL.
To address SCL function and define SCL target genes in primitive hemopoietic cells, we chose to disrupt SCL function in the CD34+/c-kit+ cell line TF-1. To this end, we took two complementary approaches. The first one consists of expressing an as-SCL that interferes with SCL protein levels, and the second one involves a dn-SCL devoid of its DNA binding domain (12), which prevents SCL function without affecting protein levels. We reasoned that loss of SCL function may be detrimental to the cells and therefore decided to analyze the cells as early as 1 wk after gene transfer, at a time when the selective pressure (G418) is already optimal. To minimize cell loss during the selective pressure and, consequently, the generation time required for in vitro expansion, we chose retroviral infection as a method for high efficiency gene delivery for which we typically obtain 30– 50% transfer efficiency. 
chain) was either similar or slightly higher. Together, the results indicate that decreased SCL protein levels (as-SCL) or decreased SCL function (dn-SCL) specifically lower c-Kit levels without affecting other surface markers.
|
We next confirmed that SCL DNA binding activity was indeed reduced in the antisense clone A31 and the dn-SCL–expressing transfectants (TF1-dn) through electrophoretic mobility shift assays using the TAL1 consensus probe (reference 21; Fig. 2). TF-1 nuclear extracts produced four specific complexes (C1–C4), as shown by self-competition (Fig. 2, lanes 11 and 12). In contrast, a probe with divergent sequences (mDE2C) did not compete for binding (data not shown), indicating that these complexes were specific. All four complexes were also found using nuclear extracts from Jurkat cells (Fig. 2, lanes 9 and 10) and TF-1 transfectants harboring the vector alone (TF1-neo, lanes 1 and 6). The identity of each complex (C1–C4) was verified by antibody supershifting. Preincubation of TF1-neo extracts with either anti-SCL or anti-E2A antibody supershifted the two most slowly migrating complexes, C1 and C2 (Fig. 2, lanes 1, 2, and 6–8), indicating that these contain SCL/E2A heterodimers as previously described (21, 24). These complexes were absent in HL-60 cells, which were included as a negative control (lane 16). In the antisense-expressing clone A31, the SCL-containing complex C1 decreased significantly, whereas the C2 complex was only marginally affected (Fig. 2, compare lanes 3 and 5), suggesting that under conditions where SCL is limiting, the smaller complex, C2, is preferentially formed over C1. Rescuing clone A31 wild-type SCL (A31-SCL) restored C1 and C2 binding to the TAL1 probe (Fig. 2, lanes 3 and 4). In all cell lines, the two fastest migrating complexes, C3 and C4, were not supershifted with anti-SCL or anti-E2A, suggesting that these did not contain the corresponding proteins. These complexes were not affected in TF1-neo but were decreased in A31, possibly as a consequence of clonal variation, since they were also decreased in A31-SCL, a subclone of A31.
|
SCL Protein Levels Specify c-kit Expression.
As observed with transient transfectants (Fig. 1), flow cytometry analysis of the A31 and TF-1 cells (Fig. 3 A) or TF1-dn and TF1-neo (data not shown) using a monoclonal antibody to human c-Kit indicated a quantitative decrease in c-Kit levels in A31 and TF1-dn as compared with control cells. There was a direct correlation with c-kit mRNA levels as determined through Northern blotting (Fig. 3 B). Thus, parental TF-1 cells or TF-1 cells harboring the vector alone (TF1-pac and TF1-neo) exhibited comparable levels of c-kit mRNA (Fig. 3 B). In contrast, c-kit expression was significantly lower in A31 and T-dn (Fig. 3, B and C). To exclude the possibility that c-kit mRNA expression was affected by the site(s) of integration in these antisense clones, we attempted to rescue c-kit expression by wild-type SCL through retrovirus-mediated gene transfer (MSCV; reference 43), using puromycin resistance as a second selective marker. Expression of the puromycin resistance gene alone did not affect c-kit expression in TF-1 cells (Fig. 3 B, TF1-pac) nor in A31 cells (data not shown). In contrast, elevating SCL expression in clone A31 restored c-kit expression (Fig. 3 B, A31-SCL) and further increased c-kit mRNA levels in parental TF-1 cells (data not shown). Together, the results indicate that the targeted attenuation of SCL protein levels and DNA binding activity directly leads to decreased c-kit expression.
|
Disrupted SCL Function Specifically Prevents SF-dependent but not GM-CSF or IL-3–dependent Cell Survival.
We have previously shown that both SF and GM-CSF suppress apoptosis in TF-1 cells. Parental cells and the different transfectants were, therefore, compared for their survival in response to optimal concentrations of SF, GM-CSF, or IL-3. Apoptotic death was revealed by the presence of a DNA ladder after agarose gel electrophoresis (data not shown). In the absence of growth factor, there was an intense DNA ladder, suggesting that the cells underwent apoptosis. At a concentration of 100 pM of SF, chosen to be near maximal suppression of apoptosis, parental TF-1 cells and the control TF1-neo line behaved similarly, with no detectable DNA degradation. Under identical conditions, apoptosis was evident in both antisense clones, A30 and A31 (data not shown), in which SCL protein levels (data not shown) and DNA binding activity (Fig. 2) were decreased. Cell viability was restored in clone A31-SCL, consistent with increased SCL DNA binding activity (Fig. 2, lane 4) and SCL protein levels (data not shown) in this clone, whereas control cells expressing the vector alone (A31-pac) behaved like the parental A31 cell line.
Cell viability was therefore quantitated using the double fluorochrome staining technique (Fig. 3 B). Full dose– response curves for SF and GM-CSF were performed in the two antisense clones, A30 and A31, as well as in control cells. Data are shown for growth factor concentrations that provide 80% survival in TF-1 cells (Fig. 3 B). As observed with agarose electrophoresis, TF-1 and TF1-neo lines remained viable with SF, whereas A30 and A31 underwent apoptosis. As above, ectopic SCL expression in clone A31 (A31-SCL) restored cell viability in SF-containing cultures, indicating that apoptotic death was due to as-SCL. In contrast, cell survival was the same in control and antisense clones in GM-CSF or IL-3 stimulated cultures (data not shown).
Apoptotic cells were also detected through Annexin V staining of membrane phosphatidyl serine, which is exteriorized during apoptotic death, both in stable clones (data not shown) and in transient transfectants 1 wk after G418 selection. Results shown in Fig. 4 indicate that parental cells and the different transfectants survive well in GM-CSF–containing cultures, consistent with their staining pattern for GM-CSF receptor (GM-R) observed in Fig. 1. In contrast, when the cells were maintained with SF, 80% of the population expressing dn-SCL or as-SCL was apoptotic, whereas both controls (TF-1 and MSCV transfectants) survived readily. Impaired survival in response to SF stimulation in these transfectants is directly correlated with decreased surface c-Kit expression as shown in Fig. 1. Together, the results suggest that SCL specifically regulates c-Kit expression and cell survival in response to SF.
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
SCL and Transcription Regulation.
The conservation of two everted E boxes at positions –381 and –374 between the human and mouse promoter sequences (49) suggests a functional role in transcription regulation, which was confirmed through binding studies (data not shown) and promoter deletion analysis. Our gel shift assays indicate that SCL-containing complexes that are formed on the c-kit probe also include E2A, consistent with the view that SCL does not bind DNA on its own and requires interaction with E12, E47, or HEB, another ubiquitous basic helix-loop-helix (21, 24). Furthermore, SCL is also found in association with the LIM-only protein Rbtn2/Lmo2 (22, 23) in a multiprotein complex with GATA-1 and a LIM-binding protein, Ldb1 (24). An E box–GATA motif was identified through in vitro binding site selection with nuclear extracts instead of isolated proteins (24), which corresponds strikingly to the c-kit everted E box sequences at –381. Sequence analysis of the human c-kit promoter also reveals the presence of several potential GATA sites at –900. Although these are found upstream instead of downstream of the E box as in the CASTing experiment, it is possible that a looping mechanism favored by protein–protein association allows for optimal spatial arrangements such as those defined by CASTing.
Transactivation assays indicate that SCL represses the E2A-dependent activity of a promoter construct containing the TAL1 motif. It is only in the presence of Id that SCL was shown to relieve the inhibition conferred by Id1 on E2A (7). In addition, despite its high affinity binding to the Ebox-GATA motif, the SCL–E2A heterodimer has no activity on a reporter construct with a minimal promoter linked to two such motives, either in the absence or presence of Lmo2. This multiprotein complex, however, provides a twofold enhancement of luciferase activity driven by GATA-1 on the same construct (24). Thus, SCL appears to be a weak transactivator, consistent with our results indicating a twofold contribution to the activity of the proximal c-kit promoter in TF-1 cells. This twofold contribution was confirmed by three distinct approaches, Northern blotting for steady state c-kit mRNA levels, flow cytometry analysis of surface c-Kit protein, and analysis of the c-kit promoter driving the luciferase reporter gene. A recent report using semiquantitative reverse transcriptase PCR did not detect significant differences in c-kit mRNA levels in embryoid bodies grown from scl–/– embryonic stem and wild-type embryonic stem cells (56). However, it should be noted that flow cytometry analysis and luciferase activity can quantitatively reveal a two- to fourfold difference, whereas reverse transcriptase PCR remains semiquantitative and requires much larger differences for detection.
Implication for Leukemogenesis.
It was previously shown that loss of SCL function is associated with premature apoptosis upon nutrient deprivation in the lymphoid cell line Jurkat (42) and decreased colony formation in the erythroid cell line K562 (53), suggesting a role for SCL in mediating cell survival. As observed with the antiapoptotic gene bcl-2, which infrequently induces lymphomas in transgenic mice after a long latency period of 18 mo (57), elevating SCL expression in thymocytes is not sufficient to cause tumors in transgenic mice (58). Directing SCL expression in the thymus can nonetheless shorten the time of appearance of T cell tumors in LMO-2 (59), LMO-1 (58), or casein kinase II transgenic mice (48). Although these observations suggest a role for SCL in leukemogenesis, its target genes remain unknown.
The cells that repopulate the thymus and a subpopulation of triple negative thymocytes are c-Kit+ (37). Interestingly, SF is expressed by fetal thymic stromal cells and epithelial cells (38, 39, 60). More importantly, T cell differentiation in thymic lobe reconstitution with fetal liver precursors is inhibited by anti-Kit (37) and is impaired in white spotting mice, indicating the functional importance of c-Kit (61). In parallel, the presence of SCL mRNA in a subset of thymocytes (11) and the absolute requirement in SCL for the generation of T cells in vivo (19, 20) are indicative of the importance of SCL for thymocyte development. Our observations suggest that SCL may also specify c-Kit expression in primitive thymocytes to sustain cell survival in response to SF. Consistent with this, elevating SCL levels in thymocytes in double CD2-SCL and CD2-Lmo2 transgenics results in a twofold expansion of the double negative CD4–/CD8– thymocyte population relative to CD2-Lmo2 transgenics (48), which, in light of our results, may be due to an increase in c-Kit+ subpopulations. We propose that constitutive SCL expression in the T cell lineage caused by chromosomal rearrangements results in constitutive c-kit expression in primitive thymocytes and prolongs their survival, which may represent an initiating event in T cell ALL. Secondary events are probably involved in the full development of T cell leukemia (58, 62), a mechanism commonly observed in tumor biology (63).
Tissue-specific Transcription Factors and Cell Survival.
The regulation of cell survival is central to normal developmental processes and stress response. It is widely accepted that survival cues are crucial for the differentiation process (64), and emerging evidence suggests a novel role for tissue-specific transcription factors, otherwise important for driving a defined pattern of gene expression, in regulating cell survival. Thus, the zinc finger transcription factor GATA-1 is required for terminal maturation in the erythroid lineage (65). GATA-1 gene ablation in embryonic stem cells blocks the differentiation at the proerythroblast stage (65, 66) and causes apoptotic death (66). Similarly, GATA-4 is required for cardiomyocyte differentiation from embryonal carcinoma cells (67) and cell survival in the cardiomyocyte pathway but not the neuronal lineage. Hence, apoptosis may be linked to insufficient survival signals, but also to inefficient or abortive cell differentiation. Similarly, SCL is required early in hemopoiesis, possibly in specifying ventral mesoderm to the hemopoietic fate (19), and is shown here to be crucial for SF-mediated cell survival. Therefore, it is proposed that lineage-specific transcription factors orchestrate both lineage-specific gene expression and the survival of the specified cell types.
|
Acknowledgments |
|---|
This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and a research traineeship from the Heart and Stroke Foundation of Canada to F. Charron. M. Nemer is a Senior Scientist of the Medical Research Council of Canada and T. Hoang is Senior Scientist from the Fonds de la Recherche en Santé du Québec and a Visiting Scientist of the National Cancer Institute (USA).
Submitted: 28 January 1998
Revised: 16 April 1998
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
1 Aplan PD, Lombardi DP, Ginsberg AM, Cossman J, Bertness VL & Kirsch IR. Disruption of the human SCL locus by "illegitimate" V-(D)-J recombinase activity, Science, 1990, 250, 1426–1429.
2 Aplan PD, Raimondi SC & Kirsch IR. Disruption of the SCL gene by a t(1;3) translocation in a patient with T cell acute lymphoblastic leukemia, J Exp Med, 1992, 176, 1303–1310.
3 Begley CG, Aplan PD, Davey MP, Nakahara K, Tchorz K, Kurtzberg J, Hershfield MS, Haynes BF, Cohen DI, Waldmann TA & Kirsch IR. Chromosomal translocation in a human leukemic stem-cell line disrupts the T-cell antigen receptor delta-chain diversity region and results in a previously unreported fusion transcript, Proc Natl Acad Sci USA, 1989, 86, 2031–2035.
4 Bernard O, Guglielmi P, Jonveaux P, Cherif D, Gisselbrecht S, Mauchauffe M, Berger R, Larsen CJ & Mathieu-Mahul D. Two distinct mechanisms for the SCL gene activation in the t(1; 14) translocation of T-cell leukemias, Genes Chromosomes Cancer, 1990, 1, 194–208.[Medline]
5 Chen Q, Cheng JT, Tasi LH, Schneider N, Buchanan G, Carroll A, Crist W, Ozanne B, Siciliano MJ & Baer R. The tal gene undergoes chromosome translocation in T cell leukemia and potentially encodes a helix-loop-helix protein, EMBO (Eur Mol Biol Organ) J, 1990, 9, 415–424.[Medline]
6 Finger LR, Kagan J, Christopher G, Kurtzberg J, Hershfield MS, Nowell PC & Croce CM. Involvement of the TCL5 gene on human chromosome 1 in T-cell leukemia and melanoma, Proc Natl Acad Sci USA, 1989, 86, 5039–5043.
7 Hsu HL, Wadman I, Tsan JT & Baer R. Positive and negative transcriptional control by the TAL1 helix-loop-helix protein, Proc Natl Acad Sci USA, 1994, 91, 5947–5951.
8 Brady G, Billia F, Knox J, Hoang T, Kirsch IR, Voura EB, Hawley RG, Cumming R, Buchwald M, Siminovitch K et al.. Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells, Curr Biol, 1995, 5, 909–922.[Medline]
9 Hoang T, Paradis E, Brady G, Billia F, Nakahara K, Iscove NN & Kirsch IR. Opposing effects of the basic helix-loop-helix transcription factor SCL on erythroid and monocytic differentiation, Blood, 1996, 87, 102–111.
10 Pulford K, Lecointe N, Leroy-Viard K, Jones M, Mathieu-Mahul D & Mason DY. Expression of TAL-1 proteins in human tissues, Blood, 1995, 85, 675–684.
11 Mouthon MA, Bernard O, Mitjavila MT, Romeo PH, Vainchenker W & Mathieu-Mahul D. Expression of tal-1 and GATA-binding proteins during human hematopoiesis, Blood, 1993, 81, 647–655.
12 Aplan PD, Nakahara K, Orkin SH & Kirsch IR. The SCL gene product: a positive regulator of erythroid differentiation, EMBO (Eur Mol Biol Organ) J, 1992, 11, 4073–4081.[Medline]
13 Begley CG, Aplan PD, Denning SM, Haynes BF, Waldmann TA & Kirsch IR. The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif, Proc Natl Acad Sci USA, 1989, 86, 10128–10132.
14 Green AR, Lints T, Visvader J, Harvey R & Begley CG. SCL is coexpressed with GATA-1 in hemopoietic cells but is also expressed in developing brain, Oncogene, 1992, 7, 653–660.[Medline]
15 Green AR, Salvaris E & Begley CG. Erythroid expression of the ‘helix-loop-helix' gene, SCL, Oncogene, 1991, 6, 475–479.[Medline]
16 Miller BA, Floros J, Cheung JY, Wojchowski DM, Bell L, Begley CG, Elwood NJ, Kreider J & Christian C. Steel factor affects SCL expression during normal erythroid differentiation, Blood, 1994, 84, 2971–2976.
17 Robb L, Lyons I, Li R, Hartley L, Kontgen F, Harvey RP, Metcalf D & Begley CG. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene, Proc Natl Acad Sci USA, 1995, 92, 7075–7079.
18 Shivdasani RA, Mayer EL & Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL, Nature, 1995, 373, 432–434.[Medline]
19 Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW & Orkin SH. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages, Cell, 1996, 86, 47–57.[Medline]
20 Robb L, Elwood NJ, Elefanty AG, Kontgen F, Li RL, Barnett LD & Begley CG. The SCL gene product is required for the generation of all hematopoietic lineages in the adult mouse, EMBO (Eur Mol Biol Organ) J, 1996, 15, 4123–4129.[Medline]
21 Hsu HL, Huang L, Tsan JT, Funk W, Wright WE, Hu JS, Kingston RE & Baer R. Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins, Mol Cell Biol, 1994, 14, 1256–1265.
22 Valge-Archer VE, Osada H, Warren AJ, Forster A, Li J, Baer R & Rabbitts TH. The LIM protein RBTN2 and the basic helix-loop-helix protein TAL1 are present in a complex in erythroid cells, Proc Natl Acad Sci USA, 1994, 91, 8617–8621.
23 Wadman I, Li J, Bash RO, Forster A, Osada H, Rabbitts TH & Baer R. Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia, EMBO (Eur Mol Biol Organ) J, 1994, 13, 4831–4839.[Medline]
24 Wadman IA, Osada H, Grütz GG, Agulnick AD, Westphal H, Forster A & Rabbitts TH. The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the Tal1, E47, GATA-1 and LDB1/NLI proteins, EMBO (Eur Mol Biol Organ) J, 1997, 16, 3145–3157.[Medline]
25 Warren AJ, Colledge WH, Carlton MB, Evans MJ, Smith AJ & Rabbitts TH. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development, Cell, 1994, 78, 45–57.[Medline]
26 Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG & Orkin SH. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells, Nature, 1989, 339, 446–451.[Medline]
27 Ono Y, Fukuhara N & Yoshie O. Transcriptional activity of TAL1 in T cell acute lymphoblastic leukemia (T-ALL) requires RBTN1 or -2 and induces TALLA1, a highly specific tumor marker of T-ALL, J Biol Chem, 1997, 272, 4576–4581.
28 Caceres-Cortes J, Rajotte D, Dumouchel J, Haddad P & Hoang T. Product of the steel locus suppresses apoptosis in hemopoietic cells. Comparison with pathways activated by granulocyte macrophage colony-stimulating factor, J Biol Chem, 1994, 269, 12084–12091.
29 Rajotte D, Haddad P, Haman A, Cragoe EJ Jr & Hoang T. Role of protein kinase C and the Na+/H+antiporter in suppression of apoptosis by granulocyte macrophage colony-stimulating factor and interleukin-3, J Biol Chem, 1992, 267, 9980–9987.
30 Williams GT, Smith CA, Spooncer E, Dexter TM & Taylor DR. Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis, Nature, 1990, 343, 76–79.[Medline]
31 Chabot B, Stephenson DA, Chapman VM, Besmer P & Bernstein A. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus, Nature, 1988, 335, 87–89.
32 Abrahamson JL, Lee JM & Bernstein A. Regulation of p53-mediated apoptosis and cell cycle arrest by Steel factor, Mol Cell Biol, 1995, 15, 6953–6960.[Abstract]
33 Copeland NG, Gilbert DJ, Cho BC, Donovan PJ, Jenkins NA, Cosman D, Anderson D, Lyman SD & Williams DE. Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles, Cell, 1990, 63, 175–183.[Medline]
34 Fleischman RA. From white spots to stem cells: the role of the Kit receptor in mammalian development, Trends Genet, 1993, 9, 285–290.[Medline]
35 Russel ES. Hereditary anemias of the mouse: a review for geneticists, Adv Genet, 1979, 20, 357–459.[Medline]
36 Li CL & Johnson GR. Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells, Blood, 1994, 84, 408–414.
37 Godfrey DI, Zlotnik A & Suda T. Phenotypic and functional characterization of c-kit expression during intrathymic T cell development, J Immunol, 1992, 149, 2281–2285.[Abstract]
38 Moll J, Eibel H, Schmid P, Sansig G, Botteri F, Palacios R & Van der Putten H. Thymic hyperplasia in transgenic mice caused by immortal epithelial cells expressing c-kit ligand, Eur J Immunol, 1992, 22, 1587–1594.[Medline]
39 Palacios R & Nishikawa S. Developmentally regulated cell surface expression and function of c-kit receptor during lymphocyte ontogeny in the embryo and adult mice, Development (Camb), 1992, 115, 1133–1147.[Abstract]
40 Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miyagawa K, Piao YF, Miyazono K, Urabe A & Takaku F. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin, J Cell Physiol, 1989, 140, 323–334.[Medline]
41 Rodriguez C, Lacasse C & Hoang T. Interleukin-1 beta suppresses apoptosis in CD34 positive bone marrow cells through activation of the type I IL-1 receptor, J Cell Physiol, 1996, 166, 387–396.[Medline]
42 Leroy-Viard K, Vinit MA, Lecointe N, Jouault H, Hibner U, Roméo PH & Mathieu-Mahul D. Loss of TAL-1 protein activity induces premature apoptosis of Jurkat leukemic T cells upon medium depletion, EMBO (Eur Mol Biol Organ) J, 1995, 14, 2341–2349.[Medline]
43 Hawley RG, Fong AZ, Lu M & Hawley TS. The Hox-11 homeobox-containing gene of human leukemia immortalizes murine hematopoietic precursors, Oncogene, 1994, 9, 1–12.[Medline]
44 Pear WS, Nolan GP, Scott ML & Baltimore D. Production of high-titer helper-free retroviruses by transient transfection, Proc Natl Acad Sci USA, 1993, 90, 8392–8396.
45 Chomczynski P & Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem, 1987, 162, 156–159.[Medline]
46 Tso JY, Sun XH, Kao TH, Reece KS & Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene, Nucleic Acids Res, 1985, 13, 2485–2502.
47 Durocher D, Chen CY, Ardati A, Schwartz RJ & Nemer M. The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium, Mol Cell Biol, 1996, 16, 4648–4655.[Abstract]
48 Kelliher MA, Seldin DC & Leder P. Tal-1 induces T cell acute lymphoblastic leukemia accelerated by casein kinase IIa, EMBO (Eur Mol Biol Organ) J, 1996, 15, 5160–5166.[Medline]
49 Tsujimura T, Morii E, Nozaki M, Hashimoto K, Moriyama Y, Takebayashi T, Kondo T, Kanakura Y & Kitamura Y. Involvement of transcriptional factor encoded by the mi locus in the expression of c-kit receptor tyrosine kinase in cultured mast cells of mice, Blood, 1996, 88, 1225–1233.
50 Therrien M & Drouin J. Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene, Mol Cell Biol, 1993, 13, 2342–2353.
51 Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST & van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis, Blood, 1994, 84, 1415–1420.
52 Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM & Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl, J Exp Med, 1995, 182, 1545–1556.
53 Green AR, DeLuca E & Begley CG. Antisense SCL suppresses self-renewal and enhances spontaneous erythroid differentiation of the human leukaemic cell line K562, EMBO (Eur Mol Biol Organ) J, 1991, 10, 4153–4158.[Medline]
54 Ohashi H, Kameda R, Nishikawa M, Kawagishi M & Liu YC. The c-kit receptor transduces the stem cell factor–triggered growth signal in murine interleukin-3– dependent cell line, Cytotechnology, 1994, 16, 27–35.[Medline]
55 Nelson BH, Lord JD & Greenberg PD. Cytoplasmic domains of the interleukin-2 receptor beta and gamma chains mediate the signal for T-cell proliferation, Nature, 1994, 369, 333–336.[Medline]
56 Elefanty AG, Robb L, Birner R & Begley CG. Hematopoietic-specific genes are not induced during in vitro differentiation of scl-null embryonic stem cells, Blood, 1997, 90, 1435–1447.
57 Linette GP, Hess JL, Sentman CL & Korsmeyer SJ. Peripheral T-cell lymphoma in lckpr-bcl-2 transgenic mice, Blood, 1995, 86, 1255–1260.
58 Robb L, Rasko JE, Bath ML, Strasser A & Begley CG. scl, a gene frequently activated in human T cell leukaemia, does not induce lymphomas in transgenic mice, Oncogene, 1995, 10, 205–209.[Medline]
59 Larson RC, Lavenir I, Larson TA, Baer R, Warren AJ, Wadman I, Nottage K & Rabbitts TH. Protein dimerization between Lmo2 (Rbtn2) and Tal1 alters thymocyte development and potentiates T cell tumorigenesis in transgenic mice, EMBO (Eur Mol Biol Organ) J, 1996, 15, 1021–1027.[Medline]
60 Wolf SS & Cohen A. Expression of cytokines and their receptors by human thymocytes and thymic stromal cells, Immunology, 1992, 77, 362–368.[Medline]
61 Rodewald HR, Kretzschmar K, Swat W & Takeda S. Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo, Immunity, 1995, 3, 313–319.[Medline]
62 Elwood NJ & Begley CG. Reconstitution of mice with bone marrow cells expressing the SCL gene is insufficient to cause leukemia, Cell Growth Differ, 1995, 6, 19–25.[Abstract]
63 Bishop JM. Molecular themes in oncogenesis, Cell, 1991, 64, 235–248.[Medline]
64 Linette GP & Korsmeyer SJ. Differentiation and cell death: lessons from the immune system, Curr Opin Cell Biol, 1994, 6, 809–815.[Medline]
65 Pevny L, Lin CS, D'Agati V, Simon MC, Orkin SH & Costantini F. Development of hematopoietic cells lacking transcription factor GATA-1, Development (Camb), 1995, 121, 163–172.[Abstract]
66 Weiss MJ & Orkin SH. Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis, Proc Natl Acad Sci USA, 1995, 92, 9623–9627.
67 Grépin C, Nemer G & Nemer M. Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcriptional factor, Development (Camb), 1997, 124, 2387–2395.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
