|28 January 1999, Volume 18, Number 4, Pages 925-934|
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|Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A|
|Patricia L Yeyati2, Rita Shaknovich2, Sima Boterashvili1,2, Jia Li2, Helen J Ball1,2, Samuel Waxman3, Kathryn Nason-Burchenal5, Ethan Dmitrovsky5, Arthur Zelent4 and Jonathan D Licht1,2,3,a|
1Derald H Ruttenberg Cancer Center, One Gustave L Levy Place, New York, NY 10029, USA
2Brookdale Center for Developmental and Molecular Biology, One Gustave L Levy Place, New York, NY 10029, USA
3Department of Medicine, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA
4Leukemia Research Fund Centre, Institute of Cancer Research, London, SW3 6JB, UK
5Laboratory of Molecular Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA
aAuthor for correspondence: Box 1130, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
The PLZF gene was identified by its fusion with the RAR locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RAR-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RAR-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.
PLZF; acute promyelocytic leukemia; cell cycle; cyclin A
Acute Promyelocytic Leukemia (APL) accounts for 10 - 15% of adult acute myeloid leukemias. Four APL-associated translocations have been described, t(15;17)(q22:q12-21) (de Thé et al., 1991), t(5;17) (q35;q21) (Redner et al., 1996), t(11;17)(q23;q21) (Chen et al., 1993) and t(11;17)(q13;21) (Wells et al., 1997), fusing the retinoic acid receptor alpha (RAR) to genes encoding PML (promyelocytic leukemia protein), nucleophosmin (NPM), the promyelocytic leukemia zinc finger (PLZF), and nuclear mitotic apparatus protein (NuMA). All four translocation products retain the DNA binding region and the ligand-dependent domain of RAR giving rise to an aberrant retinoid receptor (Chen et al., 1994; Kakizuka et al., 1991; Kastner et al., 1992; Licht et al., 1996; Pandolfi, 1996; Pandolfi et al., 1991; Redner et al., 1996). Among these molecular subtypes of APL, t(11;17)(q23;q21) APL is characterized by its poor response to all-trans-retinoic acid (ATRA) (Guidez et al., 1994; Licht et al., 1995). Translocation, t(11;17)(q23;q21) disrupts the PLZF gene yielding two new transcripts, PLZF-RAR and RAR-PLZF, both expressed in all patients tested (Grimwade et al., 1997; Licht et al., 1995). PLZF-RAR encodes an aberrant retinoid receptor which is impaired in its ability to activate RAR target genes (Chen et al., 1994; Duong et al., 1996; Licht et al., 1996). Several groups recently found that PLZF-RAR, unlike PML-RAR binds the co-repressors N-Cor and SMRT and histone deactylase 1 even in the presence of ATRA, partially explaining the ATRA-resistance of t(11;17)-associated APL (Grignani et al., 1998; Guidez et al., 1998; Hong et al., 1997; Lin et al., 1998). Nevertheless, transgenic mice harboring PLZF-RAR develop a myeloid leukemia that does respond to suprapharmacological doses of ATRA (He et al., 1998) opening the possibility that full resistance results from contributions of both PLZF-RAR and RAR-PLZF.
PLZF is a sequence-specific DNA binding transcriptional repressor differentially expressed during embryogenesis and in adult tissues (Avantaggiato et al., 1995; Cook et al., 1995). PLZF is expressed in human CD34+ cells and in multipotent progenitor cell lines such as FDCPMixA4 and at lower levels in more differentiated myeloid cell lines (Reid et al., 1995). PLZF is down-regulated as myeloid cell lines such as HL60 and NB4 are induced to differentiate with retinoic acid (Chen et al., 1993). Therefore, PLZF may play a role in the undifferentiated and quiescent phenotype of the hematopoietic progenitor (Eaves et al., 1991; Shaknovich et al., 1998).
Although binding sites for PLZF were characterized (Li et al., 1997; Sitterlin et al., 1997), until this point no target genes of PLZF were identified. We hypothesized that disruption of PLZF regulated pathways by the RAR-PLZF fusion which links the ligand-independent activation domain A of RAR to the last seven zinc finger motifs of PLZF (Li et al., 1997; Sitterlin et al., 1997) may contribute to the ATRA resistant phenotype of t(11;17)(q23;q21)-APL. Therefore, we examined the effects of engineered expression of PLZF on cell growth. We found that PLZF is a negative regulator of cell cycle progression and identified cyclin A2 down-regulation as one of the molecular events responsible for this phenotype. In contrast RAR-PLZF activated the cyclin A2 promoter, and was associated with elevated expression of cyclin A in an anchorage independent manner. Therefore cyclin A2 is a likely target gene of PLZF and aberrant gene regulation by the RAR-PLZF fusion may contribute to the aggressive phenotype of t(11;17)(q23;q21)-associated APL.
Ectopic expression of PLZF induces S phase delay/arrest
In order to investigate the primary effects of PLZF on cell cycle progression, we analysed the effect of PLZF on 32Dcl3(G/GM) murine myeloid cells (Kreider et al., 1990), representing an immortalized but non-transformed cell line. These cells were infected with a retroviral vector (Morgenstern and Land, 1990) harboring wild-type PLZF under the control of the retroviral long terminal repeat. At 48 h post infection (h.p.i.), virtually all PLZF expressing 32Dcl3 cells accumulated in the S phase of the cell cycle (Figure 1) without a concomitant increase in G2/M phase. Maintaining these cells under selective pressure in the presence of puromycin, yielded pools of cells that expressed high levels of PLZF, which accumulated in G0/G1 phase of the cell cycle, were highly suppressed in growth, and difficult to maintain in culture (Shaknovich et al., 1998). Cells stably expressing PLZF also were defective in transit into and through S phase as demonstrated by their inability cells accumulate in G2 even after 48 h of growth in the presence of nocadazole (Shaknovich et al., 1998). The growth suppressive effect of PLZF was also observed in the leukemic cell line NB4 derived from a t(15;17)-APL patient (Lanotte et al., 1991). These cells were engineered to express PLZF from an episomal vector whose copy number is controlled by selection at low levels of hygromycin (Moasser et al., 1995). Expression of PLZF in NB4 cells resulted in growth suppression (Figure 2a and b) with a higher percentage of cells in S phase compared to the controls (Figure 2c). Increased concentrations of hygromycin led to further reduction in growth rate and induced cell death of the NB4-PLZF cultures (Figure 2a) suggesting a dose dependent effect of PLZF on cell growth and cell death. PLZF also repressed growth of non-hematopoietic NIH3T3 cells (Figure 4a). Together these data indicate that PLZF is a negative regulator of cell cycle progression. The ability of PLZF to affect growth of a variety of cell lines suggested that it could affect a generic component of the cell cycle machinery.
PLZF affects cell growth by modulation of cyclin A
The negative effect of PLZF on the cell cycle (Figures 1 and 2c) suggested that PLZF may inhibit synthesis and/or activity of cyclins and/or cyclin-dependent kinases (Sherr, 1996). Among these, cyclin A2 is required for S phase entrance and transition (Girard et al., 1991; Pagano et al., 1992; Zindy et al., 1992), which suggested cyclin A2 as a candidate target gene for PLZF. Ablation of cyclin A2 expression at different points during the cell cycle resulted in blockade of DNA synthesis or cell division (Pagano et al., 1992). In transient systems, S phase arrest was associated with decreased levels of cyclin A2/cyclin-dependent kinase activity (Girard et al., 1991; Krek et al., 1995; Ogryzko et al., 1997). Furthermore, cyclin A2 expression is necessary for cell cycle progression in both normal and transformed cells, consistent with the growth suppressive effect of PLZF in all the cell lines tested. Therefore the effect of PLZF on expression of cyclin A was determined. Cyclin A levels are very low or absent in G1, begin to rise at the G1-S transition and continue to increase towards the G2/M phase (Henglein et al., 1994; Pagano et al., 1992). Hence, normally cyclin A expression would be expected to be high in S-phase cells. However, 32Dcl3 cells infected with the PLZF retroviral vector and 24 or 48 h.p.i. stained with anti-cyclin A antibodies displayed a decrease in the relative number of cyclin A positive cells compared to control cultures infected with the parental, insertless vector (Figure 3). Upon quantification, the PLZF positive cells that did express cyclin A had on average twofold less cyclin A content per cell than the controls grown under the same condition (data not shown). Furthermore, 32Dcl3-PLZF cultures in exponential growth expressed lower levels of cyclin A protein than the controls grown under the same conditions (Figure 5b, compare lanes 1 and 3). To further demonstrate the effect of PLZF on cyclin A expression, pools of NIH3T3 cells stably expressing PLZF were created (Figure 4a). As in other cell types, PLZF expression was associated with growth suppression (Figure 4b). The PLZF-expressing fibroblasts and control, vector-infected pools (Figure 4a and b) were synchronized in G0 by serum deprivation and then stimulated back into cycle by addition of 10% serum. Entrance into S phase was delayed in the PLZF-expressing pools compared to the vector control (Figure 4c). The lag in DNA synthesis correlated with delayed induction of cyclin A protein in the PLZF expressing pools (Figure 4d). Whereas control cells entered S phase in large numbers and accumulated high levels of cyclin A by 13 h after serum stimulation, cells expressing PLZF reached comparable levels of cyclin A and DNA synthesis 17 or more hours after re-entry into the cell cycle. Taken together these data indicate that PLZF inhibits expression of endogenous cyclin A, preventing and/or delaying attainment of critical levels required for normal cell cycle progression.
Cyclin A expression rescues growth suppression by PLZF
To determine the relevance of PLZF-mediated modulation of cyclin A to cell growth, the growth suppressed PLZF-expressing 32Dcl3 cells were re-infected with retroviruses harboring the human cyclin A2 cDNA (Figure 5a). No further selection was added to the super-infected cells assuming that the rescue of the growth suppression would provide selective advantage over the non infected 32Dcl3-PLZF cells. Super-infection of control 32Dcl3 cells with cyclin A2 retroviral vectors did not accelerate cell growth but restored growth to the PLZF-expressing cells (Figure 5a). Cyclin A2 overexpression was reported to be toxic in most cell lines studied (Guadagno et al., 1993; Hunter and Pines, 1991; Ohtsubo and Roberts, 1993), possibly explaining the decrease in growth of the 32Dcl3-vector pools reinfected with cyclin A2 virus (Figure 5a 32D-Vec). In contrast, cyclin A2 could be over-expressed in 32Dcl3-PLZF pools after re-infection with the retrovirus (Figure 5b, lane 2), probably because the endogenous levels of cyclin A were already reduced due to PLZF overexpression (Figure 5b, compare lanes 1 and 3).
Direct interaction of PLZF with the cyclin A2 promoter
Since PLZF is a DNA binding transcriptional repressor (Li et al., 1997), we studied the ability of PLZF and RAR-PLZF to directly interact with the cyclin A2 promoter. The human cyclin A2 promoter has two potential binding sites for PLZF, a distal site located at the nucleotide -342 and a proximal at -75 nt 5' to the major transcriptional initiation site (Figure 6a). Recombinant GST-PLZF fusion protein containing the nine zinc finger motifs of PLZF bound to both sites within the cyclin A2 promoter as determined by electrophoretic mobility shift assay (EMSA) (Figure 6b and c). When either the proximal or distal site were used as probes in an EMSA, a specific PLZF-DNA complex was formed which was competed by the presence of a molar excess of either the proximal or distal site but not the unrelated p53 binding site. Interestingly, the proximal site for PLZF in the human (Henglein et al., 1994) and mouse (Huet et al., 1996) cyclin A2 promoter overlaps with the binding site for ATF1, a transcription factor necessary to achieve maximal levels of cyclin A expression (Desdouets et al., 1996).
Since the zinc finger domains of PLZF could bind to sequences contained within the cyclin A2 promoter, we studied the effect of PLZF or the RAR-PLZF protein generated in t(11;17)(q23;q21)-APL on transcription of the cyclin A2 promoter. A construct containing nucleotides -1048/+245 of the cyclin A2 promoter and both PLZF binding sites was repressed by PLZF (Figure 7, lanes 1 and 2) as was a construct containing nucleotides -215/+245 encoding only the proximal binding site (Figure 7, lanes 4 and 5) (Henglein et al., 1994). A truncated promoter (nts -75/+100) devoid of PLZF binding sites was not repressed (Figure 7, lanes 7 and 8). Similarly RAR-PLZF did not affect transcription from a promoter devoid of PLZF sites, but in contrast activated those cyclin A2 constructs containing PLZF binding sites (Figure 7, lanes 3 and 6). We analysed the activity of cyclin A2 promoter in confluent NIH3T3 cells and observed that in spite of the low basal activity from the wild type promoter, expected under these conditions (Jansen-Durr et al., 1993) RAR-PLZF activated its transcription twofold over the control vector (data not shown). This indicated that RAR-PLZF could deregulate cyclin A expression.
RAR-PLZF deregulates cyclin A expression in vivo
Normal fibroblasts depend upon adhesion to an adequate substratum for cell cycle progression. Adhesion deprived NIH3T3 fibroblasts arrest in G1 phase (Otsuka and Moskowitz, 1975) due to down-regulation of many components of the cell cycle machinery (Zhu et al., 1996), particularly cyclin A (Guadagno et al., 1993; Schulze et al., 1995). Furthermore, growth arrest by contact inhibition is associated with cyclin A down-regulation (Yoshizumi et al., 1995) while other cyclins, such as cyclin D1 continue to be expressed (Meyyappan et al., 1998; Yoshizumi et al., 1995). As expected, control and PLZF-expressing fibroblasts (Figures 4a and 8a) expressed little or no cyclin A when grown in suspension. In contrast, cells harboring RAR-PLZF did express cyclin A protein (Figure 8). Consistent with this observation, ectopic expression of RAR-PLZF in NIH3T3 fibroblasts did not repress cell growth like wild-type PLZF and cells expressing RAR-PLZF could grow to a higher density than PLZF-expressing cells or vector controls (Data not shown). Taken together, these results suggest that in vivo, the RAR-PLZF fusion protein may uncouple cyclin A expression from adhesion and contact inhibition-dependent signals acting to stimulate cell growth (Guadagno et al., 1993).
The experiments presented above describe a biological effect of PLZF on the cell cycle and identify a target gene at least partly responsible for this phenotype. Acute, high level expression of PLZF in 32Dcl3 cells under control of the retroviral LTR led to the accumulation of cells in S phase, and a great reduction in growth rate when the infected cells were selected for stable expression in puromycin. These stable cell pools were found to reside mostly in the G1 phase of the cell cycle, were delayed in entry into S phase and were unable to traverse S phase and accumulate in G2 as expected in the presence of nocadazole (Shaknovich et al., 1998). Similarly in this report we found that PLZF suppressed growth of NIH3T3 fibroblasts. Furthermore synchronized, PLZF-expressing 3T3 cells exhibited decreased cyclin A expression and delayed entry into S phase after serum stimulation. NB4 cells stably expressing relatively low levels of PLZF also grew at a slower rate than control cells and yet exhibited an increase in the percentage of cells in S phase, consistent with a lengthening of the S phase. Taken together these data indicate that PLZF inhibits the G1/S transition and transit through S-phase through a mechanism available in a variety of cell types. This is consistent with the effect of PLZF on cyclin A, a general component of the cell cycle machinery.
The biological relevance of cyclin A as a target gene of PLZF was further underscored by the ability of a cyclin A2-expressing retrovirus to rescue the growth suppressed phenotype of 32Dcl3 cells expressing PLZF. Cyclin A itself is generally toxic to cells and did not accelerate growth of control cultures which did not express PLZF. We cannot exclude the possibility that PLZF may also affect expression of other cyclins since RAR-PLZF can modestly stimulate cell growth (PL Yeyati, R Shaknovich and JD Licht, unpublished), a phenotype also associated with cyclin D overexpression (Quelle et al., 1993). However, even if PLZF could affect expression of other cell cycle related genes, the rescue of PLZF growth suppression by cyclin A2, indicates that cyclin A2 down-regulation by PLZF is a critical molecular event underlying the ability of PLZF to repress cell growth.
The ability of PLZF to repress cyclin A expression appears to be directly at the level of transcription since PLZF can specifically bind to two sites within the cyclin A2 promoter, and can repress this promoter only when these sites are present. The expression of cyclin A in a S phase specific manner appears to be controlled by upstream activators like ATF1 and Sp1 whose activity is cyclically repressed through the action of proteins bound to more proximal promoter elements (Liu et al., 1998; Zwicker et al., 1995). The overlap of PLZF and ATF1 binding sites on the cyclin A2 promoter suggests that PLZF may compete with ATF1 binding to the promoter, retarding the achievement of maximum levels of cyclin A transcription, leading to a delayed entrance and transition through S phase. In normal blood development therefore, high level expression of PLZF might be expected to blunt the activation of the cyclin A2 promoter, thereby contributing to the quiescence of the hematopoietic progenitor cell. Down regulation of PLZF might then be accompanied by an initial increase in cyclin A and stimulation of cell growth, later to be followed by terminal differentiation. The eventual down-regulation of cyclin A observed under conditions of contact inhibition (Yoshizumi et al., 1995) or terminal differentiation (Nakamura et al., 1995) in many tissues likely results from the down-regulation of ATF1. However since PLZF may be expressed again in certain lineages such as macrophages (JD Licht and T Kalb, unpublished), the protein could potentially affect growth regulation and terminal differentiation of these cells. PLZF/ATF1 competition need not be the only mechanism of repression of the cyclin A2 promoter by PLZF as PLZF can bind to a second site within the promoter which does not overlap with an ATF1 site. Recent evidence indicates that PLZF interacts with N-Cor and SMRT, Sin3A and HDAC1, members of a multi-protein complex of histone deacetylases that is believed to lead to compaction of chromatin and a decrease in transcriptional initiation (David et al., 1998; Grignani et al., 1998; Guidez et al., 1998; He et al., 1998; Hong et al., 1997; Lin et al., 1998) even when bound at a distance from the start site of transcription.
We showed that RAR-PLZF, was an aberrant transcriptional regulator that activated, rather than repressed cyclin A2 promoter constructs containing PLZF binding sites. Our initial data also suggest that RAR-PLZF is an aberrant growth regulator. RAR-PLZF does not repress cell growth like wild-type PLZF, can be associated with increased cell growth (PL Yeyati, R Shaknovich and JD Licht, unpublished) and can immortalize murine bone marrow progenitor cells (C Lavau and JD Licht, unpublished). This could be due, at least in part to inappropriate activation of cyclin A2 transcription in vivo. Consistent with this hypothesis was the finding that RAR-PLZF activated the cyclin A2 promoter even in confluent NIH3T3 cells. Again underscoring the biological relevance of the in vitro binding and co-transfection studies, we found that fibroblasts expressing RAR-PLZF maintained expression of cyclin A in the absence of an adherent substratum, suggesting that RAR-PLZF could activate transcription of the endogenous cyclin A2 gene. We are currently investigating if RAR-PLZF is also able to support cell growth of NIH3T3 cells in an anchorage independent manner and interfere with hematopoietic differentiation. The ability of RAR-PLZF to induce cyclin A expression and to encourage cell growth suggests that RAR-PLZF may be an oncogene which plays an important role in the pathogenesis of t(11;17)(q23;q21)-associated APL. This may explain the presence of RAR-PLZF transcript in all the t(11;17)(q23;q21)-APL patients tested (Grimwade et al., 1997; Licht et al., 1995), while 30 - 40% of patients with t(15;17)-APL lack the equivalent RAR-PML transcript (Grignani et al., 1994).
These experiments identify the first candidate target gene known for PLZF, and indicate a molecular mechanism underlying PLZF growth suppression. The effect of PLZF or RAR-PLZF on endogenous cyclin A expression strongly suggests that the cyclin A2 promoter contains physiologically significant target sequences for both proteins. Furthermore, we provide evidence that RAR-PLZF is a transcription factor that may contribute to cellular transformation ultimately leading to leukemogenesis in t(11;17)(q23;q21)-APL through aberrant expression of a cell cycle control protein. In patients with t(11;17)(q23;q21)-APL, RAR-PLZF expression is driven by the retinoic acid responsive RAR2 promoter (Leroy et al., 1991). In contrast PLZF expression decreases after ATRA treatment of myeloid cells (Chen et al., 1993). Hence ATRA treatment could induce RAR-PLZF expression, potentially down-regulate endogenous PLZF and further deregulate cell growth, theoretically worsening the clinical status of patients with t(11;17)(q23;q21) associated APL. It would be of great interest to determine if RAR antagonists might, in a seemingly paradoxical manner, alleviate the clinical symptoms of t(11;17)-APL patients by down regulating RAR-PLZF expression from the RAR2 promoter.
Materials and methods
Construction of plasmids and retroviral vectors
The PLZF and RAR-PLZF cDNAs (Chen et al., 1993; Licht et al., 1996) were subcloned into the unique EcoRI or NotI site of the pBABE-puro retroviral vector (Morgenstern and Land, 1990) or into the EBOplpp episomal vector (Moasser et al., 1995). The plasmids encoding the nine zinc fingers of PLZF fused to GST, the pSG5 PLZF and RAR-PLZF (A1 domain) (Licht et al., 1996) vectors and the cyclin A2 promoter constructs (Henglein et al., 1994) were described previously. The thymidine kinase (tk) renilla luciferase reporter was purchased from Promega.
Cell culture, retroviral infection and DNA transfection
The myeloid 32Dcl3(G/GM) cell line (Kreider et al., 1990) was cultivated in IMDM supplemented with 10% heat inactivated fetal calf serum (FCS) and 5 ng/ml recombinant IL-3 (Genzyme). NIH3T3 cells were grown in DMEM with 10% calf serum (CS). The retroviral 2 packaging cell lines producing PLZF, RAR-PLZF and cyclins were maintained in DMEM/10% FCS and 293 cells were maintained in DMEM, 10% FCS supplemented with glutamine. To arrest cells in G0, NIH3T3 cells were plated at 105 cells/100 mm dish and incubated in 0.2% CS for 72 h. The cells were stimulated into cycle by the addition of fresh media containing 10% CS. For each time point of analysis several plates of cells were harvested in parallel. To analyse cyclin A expression in cells grown in suspension, NIH3T3 pools were seeded at equal densities in 10 ml of complete media onto 100 mm tissue culture plates coated with 1.5% agarose. Retroviral infections of 32Dcl3 cells were performed either by co-cultivation with the 2 producers or by incubation with undiluted fresh, filter-sterilized supernatants. Pools of retrovirus-transduced NIH3T3 cells were generated by infection with undiluted retroviral supernatants and selection for 2 weeks in puromycin (2.5 g/ml). All pools were maintained in puromycin and only early passages were used. NB4 cells, (Lanotte et al., 1991) were electroporated with the episomal vectors described and selected in DMEM containing 5% FCS and 65 g/ml of hygromycin for 3 - 4 weeks. Liposome-mediated transfection (Lipofectamine, Gibco-BRL) of 293 cells were performed as described by the manufacturer utilizing 1 g of each reporter and effector gene and 0.2 g of a tk-renilla internal control. Gene expression was assayed using the Dual Luciferase System (Promega).
Growth of viable 32Dcl3 and NB4 cells was determined by Trypan blue exclusion at the time points indicated using a Thomas' hemocytometer. Growth of triplicate cultures of NIH3T3 cells was measured by the metabolic conversion of terazolium to formazan (CellTiter96 Proliferation Assay, Promega).
Antibodies, immunoblotting and RNA blotting
Anti-PLZF monoclonal and polyclonal antibodies were generated previously (Licht et al., 1996; Reid et al., 1995). The cyclin A antibody was purchased from Santa Cruz (SC 596). For fluorescent detection of antigens anti-mouse or rabbit IgG phycoerythrin (PE) or fluorescein (FITC) conjugated secondary antibodies were used (Boehringer-Mannheim). For immunoblotting experiments, whole cell extracts were prepared as described (Licht et al., 1996) and protein concentrations were quantified using the BioRad DC Protein Assay kit. Cellular proteins were resolved by SDS - PAGE, transferred to immobilon P membranes (Millipore) incubated with anti-PLZF, or anti-cyclin A antibodies, followed by protein detection by chemoluminescence (ECL, Amersham). For RNA blot analysis, 10 g of total RNA was collected from the indicated pools and resolved through a 1.5% agarose-formaldehyde gel, transferred to a nylon filter and probed with radiolabeled PLZF cDNA as described (Chen et al., 1993).
Electrophoretic mobility shift assays
Glutathione S-transferase (GST) control or GST-PLZF proteins, purified from bacteria, were prepared as described (Ausubel et al., 1989; Li et al., 1997). Duplex oligonucleotides representing the distal or proximal PLZF site within the cyclin A2 promoter (Figure 6a) were labeled with [32P]-dCTP (3000 Ci/mM), using the Klenow fragment of E.Coli DNA polymerase I and purified using Chroma Spin Columns (Clontech). The p53 site sequence is described elsewhere and EMSA experiments were performed as described previously (Li et al., 1997).
Cell cycle analysis
For dual staining of cells for PLZF content and Propidium iodide (DNA content) or PLZF and cyclin A content, cells were permeabilized as described (Schmidt et al., 1991). For fluorescence detection of PLZF and cyclin A proteins, cells were incubated with murine, anti-PLZF and rabbit anti-cyclin A antibodies followed by incubation with species specific anti-IgG PE and FITC conjugated secondary antibodies. For cell cycle analysis, NIH3T3 cells were trypsinized and resuspended in a solution containing 0.05 mg/ml propidium iodide, 0.1% Sodium Citrate, 0.1% Triton X-100, 0.05 mg/ml RNAse DNAse free (Boerhinger-Mannheim). Data acquisition was performed with a FACScan (Becton Dickinson) flow cytometer. Since the efficiency of infection of 32Dcl3 cells was relatively low, 6´104 events were acquired in order to analyse a statistically significant number of PLZF positive cells (at least 5´103 events). Cell cycle distribution was analysed with the CellFit program (Nippon Beckton Dickinson).
We thank B Henglein, C Brechot and J Sobczak-Thepot for the cyclin A2 promoter constructs and fruitful discussions, JS Kang and RS Krauss for the cyclin-containing retroviral producer cell lines and M Serrano, N Hastie and J Caceres for careful reading of the manuscript. This work was supported by grants from the National Institutes of Health CA59936 (JDL, SW and AZ), CA62275 (ED) and the American Cancer Society grant DHP 116 (JDL). JDL is a Scholar of the Leukemia Society of America. PLY was supported by the Lauri Strauss Leukemia Fund. RS was supported by a Medical Scientist Training Grant GM0707280-17 from the NIH. KN-B was supported by National Research Service Award CA61646. HJB was supported by the Leukemia Research Foundation. This is publication # 250 from the Brookdale Center for Developmental and Molecular Biology.
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Figure 1 Ectopic expression of PLZF induces S phase arrest/delay. 48 hours after infection with PLZF-encoding retrovirus, 32Dcl3 cells were permeabilized and stained with anti-PLZF monoclonal antibody and propidium iodide for DNA content. An uninfected control was similarly stained. PLZF expression is plotted on the Y axis and DNA content along the X axis. PLZF positive cells lie above the horizontal bar indicating basal fluorescence of the isotype control antibody. The cell cycle distribution for each sub-population is shown on the right
Figure 2 PLZF inhibits growth of NB4 APL cells. (a) NB4 cells were electroporated with control or PLZF episomal vector and selected in 65 g/ml hygromycin for 4 weeks. After this primary selection, the cultures were divided and grown in 65 or 180 g/ml hygromycin. The number of live and dead cells was determined by trypan blue exclusion. (b) Immunoblot analysis of PLZF expression in NB4 cells grown in 65 g/ml of hygromycin. (c) The DNA content of exponentially growing cultures of NB4 cells expressing PLZF was determined by flow cytometry. The bars represent percentage of cells in G0/G1, S and G2/M respectively. The data represent the average and standard deviation of four independent experiments
Figure 3 PLZF decreases cyclin A protein expression in hematopoietic cells. 32Dcl3 cells were co-cultured with PLZF and vector retroviral producer cell pools and fed fresh media 24 h post infection (hpi). At 24 or 48 hpi, cells were permeabilized, incubated with anti-cyclin A primary antibody and anti-IgG-PE-conjugated secondary antibody and analysed by flow cytometry. Cyclin A negative cells were selected based on the PE intensity of cells stained with an isotype control antibody. The percentage of events in each region was calculated and the Y axis represents the ratio of cyclin A positive to cyclin A negative cells from a representative experiment. Black bars represent PLZF-infected cells and shaded bars vector infected 32Dcl3 cells
Figure 4 PLZF expression delays entry into the cell cycle and induction of cyclin a protein. (a) Growth curves of triplicate cultures of three independent stable cell pools of NIH3T3 cells generated by retroviral infection with PLZF encoding retroviruses or an empty retroviral vector as determined by metabolic conversion of tetrazolium into formazan. (b) Immunoblot of whole-cell lysates from the stable pools with anti-PLZF antibody, lane 1: Vec-1, lane 2: PL1, lane 3: PL-2 lane 4: PL-3. (c): Puromycin-resistant NIH3T3 stable pools harboring PLZF (PL-1, PL-2 and PL-3) or the control retrovirus (Vec-1) were synchronized in G0 by serum deprivation for 72 h and were re-stimulated into the cell cycle with 10% serum. At the indicated times post-refeeding, the cultures were examined for DNA content by propidium iodide staining and flow cytometry. The percentage of cells in S phase is plotted versus time after serum stimulation. (d) Whole cell lysates (50 g) from NIH3T3 cells harvested at the time points indicated in the experiment described above were immunblotted with anti-cyclin A antibody
Figure 5 Ectopic expression of human cyclin A2 reverses growth suppression by PLZF. (a) Growth curves of puromycin-resistant 32Dcl3-PLZF or vector control pools. 32Dcl3 cells were infected in parallel with a vector control virus or a virus encoding PLZF and stably selected as puromycin resistant cultures. The stable pools were super-infected for 48 h in separate experiments with undiluted supernants containing retrovirus encoding human cyclin A2 or an insertless control retrovirus. Three to seven days after re-infection the cells were seeded at 104 cells/ml and live cells were counted daily by Trypan blue exclusion. All cultures were maintained in puromycin and no new selective pressure was added after re-infection. The original pools were: 32D-Vec (vector control) and 32D-PL7 (PLZF expressing pool). These pools were super-infected with: , cyclin A2 retrovirus; insertless retrovirus. (b) Cyclin A2 levels are restored in PLZF expressing cells. Immunoblot of lysates from PLZF7 cells or the control vector before and after infection with cyclin A2 retrovirus. Lane 1: 32D-PLZF7 pool uninfected, Lane 2: 32D-PLZF7 pool-infected with cyclin A retrovirus, Lane 3: 32Dcl3 control vector cells without secondary infection, Lane 4: 32Dcl3 control vector cells, infected with cyclin A retrovirus
Figure 6 PLZF protein directly interacts with the cyclin A2 promoter. (a) Schematic depiction of the human cyclin A2 promoter (adapted from (Desdouets et al., 1996)) and the binding sites for PLZF. The +1 position corresponds to the major initiation site of transcription. The distal and proximal binding sites for PLZF in the promoter are shown, upper case letters from these sites denote the nucleotides common among the cyclin A2 promoter and the sequences described as binding sites for PLZF (Li et al., 1997). (b, c) In vitro binding of GST-PLZF to sites within the cyclin A2 promoter. Radiolabeled synthetic duplex oligonucleotides corresponding to the distal (b) or proximal (c) PLZF binding sites shown in a were incubated with purified GST-PLZF protein in the presence or absence of a 100-fold molar excess of unlabeled competitor as indicated. The complexes were resolved through 6% (b) or 8% (c) non-denaturing polyacrylamide gels. Lane 1: probe alone, lane 2: control GST protein, lanes 3 - 6: GST-PLZF protein, lane 3: no competitor, lane 4: p53 binding site competitor, lanes 5 and 6 unlabeled competitors containing either the proximal or distal PLZF sites of the cyclin A2 promoter
Figure 7 PLZF and RAR-PLZF affect transcription of the cyclin A2 promoter. Reporter plasmids containing different portions of the human cyclin A2 promoter transfected in triplicate into 293 cells along with either pBABEpuro-PLZF or the parental pBabepuro vector or pSG5-RAR-PLZF and pSG5 vector as effectors. A thymidine kinase-renillin luciferase reporter was included to determine the efficiency of transfection; only those transfections with comparable renillin expression were analysed. The values represent luciferase expression (±standard deviation) relative to control vectors without inserts. The effectors were transfected in the following order: Lanes 1, 4 and 7: vector control, lanes 2, 5 and 8: PLZF expression vector, lanes 3, 6 and 9: RAR/PLZF expression vector. Several different genomic fragments from the cyclin A2 promoter were used as reporters: lanes 1 - 3: (-1048/+245) reporter, lanes 4 - 6: (-215/+245) reporter, lanes 7 - 9: (-75/+100) reporter
Figure 8 RAR-PLZF deregulates cyclin A expression in anchorage-deprived fibroblasts. (a) Northern blot analysis for PLZF and RAR-PLZF expression in NIH3T3 cells. Each lane contains 10 g total RNA from: lane 1, parental NIH3T3 cells; lanes 2 and 3: PL-2 and PL-1 (PLZF pools 2 and 1), lanes 4 and 5: Vec-2 and Vec-1 (vector pools 2 and 1), lanes 6 and 7: R/P-2 and R/P-1 (RAR-PLZF pools 2 and 1). (b) Cyclin A levels of stable pools of NIH3T3 grown on agarose coated plates for 72 h. Whole cell lysates from NIH3T3 pools expressing PLZF or RAR-PLZF were immunoblotted with anti-cyclin A antibodies. Experiment 1: Lanes 1 - 3: three independent pools of NIH3T3 stably expressing PLZF; lane 4: NIH3T3 harboring an insertless control retrovirus, lane 5: NIH3T3 cells stably expressing RAR-PLZF. Experiment 2: lane 6: control NIH3T3 cells, lanes 7 and 8 Pools of NIH3T3 cells stably expressing RAR-PLZF
|Received 29 May 1998; revised 18 August 1998; accepted 18 August 1998|
|28 January 1999, Volume 18, Number 4, Pages 925-934|
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