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Leukemia Research 30 (2006) 437–447
Expression of PDZ-binding kinase (PBK) is regulated by cell cycle-specific transcription factors E2F and CREB/ATF Asit K. Nandi ∗ , Aaron P. Rapoport University of Maryland School of Medicine, Greenebaum Cancer Center, 655 W Baltimore Street, Baltimore, MD 21201, USA Received 3 June 2005; received in revised form 9 August 2005; accepted 11 August 2005 Available online 19 September 2005
Abstract Earlier we reported that a novel mitotic protein kinase, PDZ-binding kinase (PBK), is expressed in primary hematopoietic neoplasms. Recent reports have suggested a role for PBK in mitotic progression. In the present study, we demonstrate that PBK is downregulated during doxorubicin induced growth arrest of HL60 promyelocytic leukemia cells at least partly due to cell cycle-specific transcriptional regulation. Furthermore, we show that transcriptional control is mostly due to binding of transcription factors E2F and CREB/ATF to two distinct binding sites within the PBK promoter. This was demonstrated by: (i) electrophoretic mobility shift assays showing transcription factor binding within the PBK promoter at the putative E2F (−146 bp) and CREB/ATF (−312 bp) binding sites; (ii) Western immunoblot analysis of knockdown extracts from siRNA inhibition of transcription factor expression showing that PBK protein expression is dependent upon the presence of these transcription factors; (iii) codistribution of CREB factor and PBK in cell lines of disparate tissue origin; and (iv) luciferase reporter assays showing that PBK promoter activity is dependent on factor binding at intact E2F and CREB/ATF sites. These findings may provide insight into the mechanisms that upregulate PBK expression in proliferative hematologic malignancies and downregulate its expression following growth arrest of leukemic cells. © 2005 Elsevier Ltd. All rights reserved. Keywords: PDZ-binding kinase; Cell cycle arrest; Transcription factors; E2F; CREB/ATF; Leukemic cells; PBK/TOPK
1. Introduction PDZ-binding kinase (PBK) or T cell originated protein kinase (TOPK) is a serine–threonine kinase, which is homologous to MAP kinase kinase MKK3 and has been reported to be activated during mitosis [1,2]. PBK was identified in our laboratory as an expressed sequenced tag (EST), which was differentially expressed in the Burkitt’s lymphoma cell line GA10 compared to hyperplastic tonsillar B cells [3]. PBK has been cloned as an interaction partner of the human tumor suppressor Discs-large (hdlg) by yeast two-hybrid screening of a HeLa cell cDNA library [1]. Other investigators cloned PBK as an IL-2 inducible gene in cytotoxic T lymphocytes and by protein–protein interactions with human C-Raf [2,4]. ∗
Corresponding author. Tel.: +1 410 328 1906; fax: +1 410 328 6559. E-mail addresses: [email protected] (A.K. Nandi), [email protected] (A.P. Rapoport). 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.08.011
The murine homologue was cloned as an IL-6 stimulated gene from myeloma cells [5]. PBK mRNA is expressed in a wide variety of tumor cell lines and in normal testicular and embryonic tissues [3]. Previous studies have shown that PBK protein is also expressed in primary clinical samples from patients with acute myelogenous leukemia (AML) and aggressive lymphoid neoplasms [6]. In contrast, PBK expression was barely detectable in CD34+ immunoselected peripheral blood progenitor cells. Consistent with previous findings, our data indicated that PBK phosphorylates p38 MAP kinase, thereby placing PBK as a MAP kinase kinase in the signal transduction pathway [7,8]. However, it is likely that other as yet unidentified cellular targets of PBK exist [9]. PDZ-binding kinase is activated possibly by Cdc2cyclinB during the mitotic phase [1]. Consequently, PBK may be a mitotic regulator. Similar to protein kinases PLK, Aurora kinases and NIMA, PBK may have a role in progres-
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sion through the mitotic phase [10–12]. In a recent report PBK/TOPK has been shown to be associated with mitotic spindles through a protein phosphorylation site recognized by Cdc2-cyclinB. Further studies involving knockdown RNA expression have suggested that PBK has a role in cytokinesis [9]. Studies using HL60 promyelocytic leukemia cells have shown strong downregulation of PBK protein expression after TPA-induced differentiation along the macrophage pathway [6,13,14]. Taken together, these data suggest that PBK expression may help regulate leukemic cell growth or survival. When exposed to genotoxic stress, many normal as well as tumor cells undergo cell cycle checkpoint activation and cell growth restriction in the G1 and G2 phases of the cell cycle [15–18]. Several regulators including p53 and p14ARF have a role in implementing the cell cycle blocks [19–21]. Downregulation of two mitotic regulators Stathmin and AIM1 following induction of G2/M arrest by the antineoplastic drug doxorubicin has been reported to be mediated by E2F factor [22–24]. A decrease in the transactivation function of the CREB factor also takes place in response to genotoxic stress [25–27]. Induction of cellular senescence by doxorubicin has been studied by several investigators [28–30]. Here we have examined the regulation of PBK during cellular senescence of HL60 promyelocytic leukemia cells following exposure to doxorubicin. We have found that PBK protein expression is downregulated during growth arrest by doxorubicin. We also demonstrate that the transcription factors E2F and CREB/ATF are critical regulators of PBK expression during growth arrest. These findings may have implications for the study of leukemic cell growth and survival.
2. Experimental procedures 2.1. Cell growth arrest and Western blot analysis Doxorubicin (Sigma) was dissolved in sterile water at a concentration of 2 mg/ml and filtered through 0.8/0.2 m syringe filters. The stock was diluted 1:10 (1000×) and stored at 4 ◦ C. HL60 cells were resuspended to a density of 4–6 × 105 cells/ml in the presence of 0.2 g/ml doxorubicin and maintained in the CO2 incubator at 37 ◦ C for varying periods of time. Aliquots of cells were taken out at specific time points, pelleted and resuspended in sterile PBS. Cells were lysed in 60 l CelLyticTM buffer (Sigma) containing protease inhibitors. The protein contents were measured using the BCA protein assay kit (Pierce) and aliquots containing 50 g of protein were run on 10% Tris-glycine gels and transferred to PVDF membranes according to the manufacturer’s protocol (Invitrogen). Membranes were blocked with ready to use blocking agent (KPL) for 1 h at 25 ◦ C and probed with a monoclonal antibody to PBK (BD Pharmingen) in the same blocking buffer for overnight at 4 ◦ C. The blots were processed according to standard methods and crossreacted with a HRP-linked secondary antibody (Amersham), washed
and developed using ECLTM detection reagents according to the supplier’s protocol (Amersham). Other antibodies utilized included actin antibody (Sigma), phospho-Cdc2 Tyr15 (cell signaling technology), and phospho-c-Myc antibodies (Cell Signaling Technology) all of which were blocked with 5% nonfat milk in TBST buffer. TranSilentTM Western blots containing lysates of human embryonic kidney 293 cells following knockdown of various transcription factors, were obtained from Panomics. Aphidicolin (Calbiochem) was dissolved at a concentration of 0.33 mg/ml in 70% ethanol and was used for S phase arrest at a final concentration of 3 g/ml for 24 h. Following this period, cells were washed in sterile PBS and suspended in prewarmed growth medium. 2.2. Genomic amplifications and construction of luciferase expression plasmids Genomic DNA was isolated from HL60 cells using High Pure PCR Template Preparation kit (Roche) and amplified using primers derived from the putative promoter of the PBK gene (5 -GCGCTAGCCTGAATTGCTGATGTTACAGG-3 , 5 -GCAGATCTCCTCTAGCACCAACACATACG-3 ) and a high fidelity DNA polymerase mix (Roche). BglII and NheI restriction sites were inserted in the primers. The cycling conditions were 95 ◦ C-1 , 51 ◦ C-3 , 68 ◦ C-2 for 35 cycles with an initial denaturation of 2.5 at 95 ◦ C and an extension period of 10 at 68 ◦ C at the end. The amplified 2.5 kb band, was gel purified and cloned into the PCRII TOPO T/A cloning vector (Invitrogen). The sequence of the amplified piece was confirmed following isolation of transformant clones derived from TOP10 cells. The BglII-NheI restriction fragment containing the 2.5 kb promoter in the sense orientation was cloned next to a luciferase coding sequence in the pCBRBasic vector (Promega). The 2.5 kb promoter construct in the pCBR-Basic vector was digested with KpnI to generate a 2.0 kb distal promoter fragment. The large Kpn fragment containing the rest of the plasmid including the 0.6 kb ATGproximal promoter piece next to the luciferase coding unit was also generated through religation. The 2.0 kb distal promoter was subsequently cloned next to the luciferase coding region in the sense orientation. Thus, two luciferase constructs were generated which contained the ATG-proximal promoter sequence from +27 to −558 bp position and the distal promoter sequences from −559 to −2498 bp position (with respect to the transcription start site). 2.3. RT-PCR In order to measure the level of PBK mRNA following doxorubicin treatment, total RNA was isolated at specific time points from 40 ml cultures using TrizoleTM reagent (Invitrogen). Aliquots equivalent to 2 g of total RNA were reverse transcribed and PCR amplified using onestep RT-PCR (Invitrogen) in the presence of two pairs of primers—one for amplification of the entire PBK coding
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region (5 -GCGGATCCCTTCCAGGCGGTGAGACTCTGGACT-3 and 5 -GCGTCGACGACATCTGTTTCCAGAGCTTCAAC-3 ) and the other for amplification of GAPDH as a control for constitutive gene expression (Q.Biogene). Conditions for thermal cycling were 50 ◦ C for 30 and 94 ◦ C for 2 followed by 30 cycles at 94 ◦ C-30 , 55 ◦ C-1 , 68 ◦ C-2’ and a final extension time of 7 at 72 ◦ C. 2.4. Electrophoretic mobility shift assay A pair of complementary 34-mers (5 -CCTATAATAAAATTGACGTCATAATCACGACACA-3 ) overlapping to the putative CREB-binding core motif ‘TGACGTCA’ present in the PBK promoter, were synthesized, annealed by slow cooling from 95 to 25 ◦ C and labeled using Digoxygenin11ddUTP following the manufacturer’s protocol (Roche). Gel shift assay for the CREB factor was carried out utilizing DIG-Gel Shift Kit (Roche) and 10 g of HeLa (Promega) or Raji (Active Motif) nuclear extracts. The complex was formed in the presence of poly[d(A–T)] as a nonspecific competitor and was resolved in precast 5% native polyacrylamide gel (Invitrogen) prerun for 1/2 h using 0.5 × TBE running buffer at 4 ◦ C. The E2F gel shift was carried out using a double stranded oligomer (5 -TTCCTTCCCTCGTCTTTGGCGCCTGCGGGCACCGGGAGT-3 ) and poly[d(IC)] as a nonspecific competitor in Raji and human embryonic kidney (HEK) 293 nuclear extracts (Active Motif). The gel run was carried out at 4 ◦ C for 2 h in 0.25× TBE buffer at 100 V. Following electrophoresis, the gels were blot transferred to positively charged nylon membranes utilizing the semidry blotting procedure, UV crosslinked and air dried. The membranes were subsequently developed following a chemiluminescent detection technique as specified by the manufacturer (Roche). To demonstrate the effect of antibodies towards the formation of specific gel shift complexes, nuclear extracts were preincubated with phospho-CREB (Ser133), phospho-ATF2 (Thr71) (Cell Signaling Technology) and E2F1 (Upstate) antibodies. 2.5. Luciferase assay HEK 293 cells (6–8 × 105 ) were plated in 60 mm tissue culture dishes 24 h before transfection. On the day of experiment, cells were fed with fresh medium 3–5 h prior to transfection. Two micrograms of the designated luciferase plasmid construct was mixed with 2.0 g of control betagalactosidase expression plasmid in a total volume of 25 l and were combined with 15 l FuGene6TM (Roche) and 100 l DME (no serum) according to protocol. The mixture was allowed to incubate for 15 min at room temp and was added to the cells without changing medium. At 48 h post-transfection, cells were harvested by scraping using sterile PBS and lysed in 250 l Glo-lysisTM buffer (Promega). The beta-galactosidase and luciferase assays were carried out using Beta-GloTM and Chroma-GloTM luciferase assay sys-
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tems (Promega) utilizing 2.5 l of 1:20 diluted extracts and 100 l of original extracts, respectively. Both luciferase and beta-galactosidase enzyme activities were measured in the TD 20/20 luminometer manually. 2.6. Site-directed mutagenesis The 0.6 kb proximal promoter construct was utilized to carry out site-directed mutagenesis of the putative E2F binding site following the quick change site-directed mutagenesis protocol (Stratagene). A pair of complimentary 33-mer oligonucleotides incorporating mutant bases (5 -CCTTCCCTCGTCTTTGTATCCTGCGGGCACCGG-3 ) were mixed with 10 ng of circular plasmid DNA in the appropriate buffer in the presence pfu ultra HF DNA polymerase and incubated in the thermal cycler programmed to undergo 20 cycles of 95 ◦ C-30 s, 55 ◦ C-1 min, 68 ◦ C-5 min following an initial denturation at 95 ◦ C for 30 s. The mixture of input and amplified DNA was digested directly with DpnI to eliminate parent DNA molecules and was transformed into XL-1 Blue cells. The mutant plasmids were indicated by loss of a NarI restriction cleavage site and further confirmed by sequencing using primers specific for the luciferase vector (Reporter Vector Primer3, Promega). Mutagenesis at the putative CREB binding site was carried out in the original PCRII TOPO vector with the entire 2.5 kb promoter fragment by deletion of four nucleotide 3 overhang ends following AatII digestion utilizing T4 DNA polymerase in the presence of dNTP [31]. The AatII restriction site was located within the core motif for CREB/ATF binding. Following confirmation by sequencing, the BglII/KpnI piece covering the 0.6 kb promoter along with the CREB site mutation was cloned into the pCBR-basic luciferase vector in the sense orientation. 2.7. Flow cytometry 1–2 × 106 Cells at specific time points were withdrawn from appropriately treated cultures, diluted with PBS (4 ◦ C) and were pelleted. Cells were resuspended thoroughly in 1 ml PBS, 2 ml 100% ethanol (−20 ◦ C) was added dropwise while shaking and the cell suspension was stored at −20 ◦ C until ready for flow cytometry. Two or three hours before analysis, cells were pelleted at 2000 rpm, suspended in 1 ml PBS, allowed to stand for 5 min, and were finally resuspended thoroughly in 1 ml propidium iodide-staining solution with RNase (BD Pharmingen) for 30 at 25 ◦ C. Cells were subsequently analyzed in FACScan (Becton Dickinson Instruments) utilizing the Cell Quest program. 3. Results 3.1. PBK protein is downregulated during growth arrest We first examined PBK expression in HL60 cells following treatment with doxorubicin. A variety of tumor cell
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Fig. 1. Doxorubicin induced cell cycle arrest of leukemic cells. The HL60 promyelocytic leukemia cells were treated with 0.2 g/ml doxorubicin (Dox), harvested after 0, 3, 6, 9, 12, 24 and 48 h as indicated and analyzed in the FACScan flow cytometer following RNase digestion and propidium iodide staining. M1, M2 and M3 represent peaks corresponding to the G1, S and G2/M phases, respectively. Only the G2% values are shown. The subdiploid population reflects less than 20% trypan blue positive cells, which did not change significantly during the course of the experiment.
lines, which do not carry a functional copy of the p53 gene undergo growth arrest in the G2/M phase in the presence of low doses of doxorubicin and the cells acquire senescencelike phenotype (SLP) [22,28–30]. Fig. 1 shows initiation of rapid cell growth arrest predominantly at the G2/M phase in the presence of 0.2 g/ml of doxorubicin. To further document growth arrest, the phosphorylation status of the Tyr 15 residue of the mitotic cyclin, cyclin B-regulated-cyclin dependent kinase (Cdc2) was examined. Phosphorylation of this site is carried out by Wee1 kinase which negatively regulates Cdc2 activity, while the Cdc25C phosphatase causes dephosphorylation of the Tyr15 residue. A decrease in the phosphorylated form of Cdc2 may indicate cell cycle arrest at the G2/M boundary [32–34] (see Section 4). As shown in Fig. 2, the level of Tyr 15 phosphorylation of Cdc2 decreased rapidly to an undetectable level by 48 h. While the HL 60 cells were growth-arrested, PBK expression diminished gradually at 48 and 72 h and at about 96 h post-treatment, PBK became nearly undetectable. The slow disappearance kinetics of PBK in this system is consistent with our previous observation regarding the rate of PBK disappearance after TPA induction of HL60 cells [6]. The level of phosphorylated (Ser780) retinoblastoma protein (Rb) remained constant for most of the time period and was slightly upregulated at 96 h and the level of phosphohistone-H3 (Ser10) remained constant (data not shown) indicating that there was no significant indiscriminate protein degradation. Actin levels also did not change during doxorubicin induction. In addition, cell viability was not affected significantly by doxorubicin treatment (80–90% viable cells by trypan blue exclusion).
Fig. 2. Downregulation of PBK protein level during growth arrest. Western immunoblot analysis of HL60 cell lysates after 0, 24, 48, 72 and 96 h of doxorubicin (0.2 g/ml) treatment. PBK was detected using a monoclonal PBK antibody. P-Cdc2 represents the levels of the Tyr15 phosphorylated form of Cdc2, a cyclin B dependent kinase at different timepoints. Phosphoc-Myc represents the level of the Thr58/Ser62 phosphorylated forms of cMyc protein. Stable actin and phospho-Rb levels are shown to demonstrate cell cycle arrest in the absence of generalized protein degradation. The minus (−) and plus (+) lanes represent control and apoptotic samples, respectively for Jurkat cells treated with camptothecin, to demonstrate that phospho-Rb disappears during apoptosis.
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We reported previously that phosphorylated c-Myc is downregulated during TPA induced differentiation of HL60 cells prior to PBK downregulation and that PBK was coexpressed with activated c-Myc in a number of human leukemias and lymphomas [6]. Therefore, c-Myc expression was monitored in the doxorubicin treated cells. Fig. 2 shows that downregulation of the phosphorylated form of c-Myc protein occurred prior to PBK downregulation. These results are in accordance with previous findings suggesting that PBK expression is correlated to active cellular growth and conditions which favor departure from the active growth phase either by inducing terminal differentiation or proliferation arrest, cause diminished expression of PBK. Thus, the c-myc gene being well known for its response to mitogenic or growth inhibitory stimuli, is co-regulated with PBK expression in these two models [35]. 3.2. Regulation of PBK involves cell cycle specific mRNA accumulation In order to examine whether PBK downregulation during growth arrest occurs at the transcriptional level or is caused by selective protein degradation, we analyzed PBK mRNA level by quantitative RT-PCR. Total RNA was isolated at different time points during doxorubicin treatment from HL60 cells, reverse transcribed and the cDNA was amplified in a single step in the presence of two sets of primers for PBK and GAPDH. The expected product for the GAPDH amplification was 516 bp while the other pair of primers amplified a 1 kb fragment covering the PBK coding region. As shown in Fig. 3A PBK mRNAs were maintained at a low level in asynchronous cells. After doxorubicin treatment, there was apparent upregulation possibly due to synchronization of cells in the G2 phase of the cell cycle within a period of 24 h followed by down regulation in the subsequent period as the cells become growth arrested. The 516 bp GAPDH product remained constant indicating that the amplification had been quantitative. These results indicate that PBK is negatively regulated at the level of mRNA following sustained cell cycle arrest of HL60 cells.
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PBK expression was further examined following synchronization of HL60 cells in the presence of the reversible DNA polymerase inhibitor aphidicolin and allowing resumption of cell cycle propagation. The level of PBK mRNA was significantly higher at the G1 phase (46.2% G1 at 0 h) compared to asynchronous cells and dropped when cells were predominantly in S phase (40.2% S at 4 h) (Fig. 3B). The level of PBK mRNA was again higher when cells were predominantly in the G2 phase (31.7% G2 at 8 h). At 12, 16 and 20 h the transcript levels maintained a similar profile. The levels of amplified GAPDH cDNA were nearly constant between 0 to 20 h. These results indicate that PBK mRNA accumulation is cell cycle dependent. These results in the HL60 cells are consistent with HeLa cell cycling studies involving microarray analysis, which show that PBK mRNA is cell cycle regulated [36]. In addition, a recent report by Matsumoto et al. also demonstrated that PBK/TOPK protein expression exhibits cell cycle specific regulation [9]. 3.3. PBK promoter encompasses binding sites for CREB/ATF and E2F transcription factors To study the mechanisms which regulate transcription, a 2.5 kb putative promoter for the PBK gene was amplified utilizing database sequence and genomic amplification. Human genomic DNA isolated from HL60 cells was amplified using primers designed to incorporate NheI and BglII restriction sites at the 5 and 3 ends of the promoter, respectively. On analysis, the PBK promoter did not contain a canonical ‘TATA’ box, but instead an A–T rich sequence occurred around −30 position with respect to the RNA start site [2,37]. Thus, PBK belongs to the class of ‘TATA’ less genes. We first determined if the PBK promoter region contained known transcription factor binding sites which could be implicated in cell cycle regulation. Based upon scanning for consensus transcription factor binding motifs, several putative transcription factor binding sites were localized within the 2.5 kb PBK promoter. We focused on two of the factor binding sites in the proximal part of the promoter. Thus, putative consensus binding sites were identified for E2F and CREB/ATF factors at positions −146 bp and −312 bp with respect to the RNA
Fig. 3. Cell cycle specificity of PBK mRNA accumulation by RT-PCR analysis. Total RNA was isolated from HL 60 cells following: (A) growth arrest by doxorubicin (0.2 g/ml) (DOX) and (B) cell synchronization in the presence of aphidicolin (3 g/ml) (APHI) for 24 h and subsequent growth in drug free medium for the designated periods. RNA analysis was carried out by one step RT-PCR and subsequent gel electrophoresis on agarose gels. The 1 kb amplification product was derived from PBK mRNA and the 516 bp product from GAPDH mRNA, respectively and have been identified. The PBK and GAPDH bands shown were derived from the same experiment.
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Fig. 4. Nucleotide sequence of a segment of the putative PBK promoter. The promoter sequences upstream of the PBK mRNA coding region has been derived from the Homo sapiens chromosome 8 genomic contig NT 007988 [gi:27478833]. Nucleotide position ‘+1 refers to the transcription start site corresponding to the 5 nucleotide of the PBK messenger RNA (1). The putative CREB/ATF and E2F binding sites have been shown. The underlined sequence represents an A/T rich cluster in place of a canonical TATA box.
start site for further study(Fig. 4). The selection of E2F and CREB/ATF was also motivated by the previous observation, which indicated that PBK was cell cycle regulated. Electrophoretic mobility shift assays were performed to determine if transcription factors in the nuclear extracts actually bind to the putative sites identified in the PBK promoter sequences, as stable DNA–protein interactions require further flanking sequences besides the core binding motifs. By electrophoretic mobility shift assay (EMSA) we detected fac-
tor binding at the CREB/ATF site using a HeLa cell nuclear extract (Fig. 5A) and also Raji nuclear extract (Fig. 5B). These cell lines were selected for analysis because they express the PBK protein at a high level. So, the key transcription factors, which regulate PBK gene expression should be present in these extracts. Binding was partially inhibited by both CREB antibody and ATF2 antibody but not by a heterologous E2F antibody indicating that CREB factor binds at the −312 bp position within the PBK promoter in the HeLa cell extract. Multiple complexes in the Raji nuclear extract possibly represent different combinations of CREB related factors that bind as homo-or heterodimers. A number of cell lines derived from multiple tissues of human, rat and mouse origin that were not previously analyzed were tested to determine if expression of transcription factor CREB coincides with PBK expression. Fig. 6 shows a Western blot analysis of CREB and PBK expression in the following cell lines: human fibroblasts (HS68), fibrosarcoma (HT1080), T cell leukemia (Jurkat) as well as mouse embryonic fibroblasts (C3H10T1/2), embryonal carcinoma (F9) and rat skeletal muscle myoblast (L6). Antibodies which crossreact with human, mouse and rat antigens were used and it was found that the two cell lines, Jurkat and F9 cells, which expressed PBK also expressed detectable levels of CREB factor. Notably, PBK was expressed at a low level in F9 cells which also expressed a low level of CREB factor, while the other lines had no detectable expression of either protein. Considered together these results suggested that CREB factor may regulate PBK expression in vivo.
Fig. 5. Gel mobility shift assay to demonstrate CREB/ATF factor binding within the PBK promoter at −312 bp position. A pair of double stranded 34 bp oligomers were derived from the PBK promoter encompassing the putative CREB binding site. These were labeled with Digoxygenin 11-ddUTP and were tested by the electrophoretic mobility shift assay using the HeLa nuclear extract (A) and Raji nuclear extract (B). Poly[d(A–T)] was used as a nonspecific competitor in this assay. A major band which is competed by the homologous unlabeled oligomers (“Homolog Comp”) has been identified as a specific gel shift complex. As expected a mutant competitor (“Mutant Comp”) showed no inhibitory activity in the Raji nuclear extract assay.
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E2F factor binds at the expected site within the PBK promoter. As shown in Fig. 7B, the faster migrating complex was not competed by unlabeled homologous competitor and was considered to be nonspecific. The slower migrating complex however demonstrated specific competition but was not affected by the addition of E2F1 antibody. Lack of interference by the presence of specific antibody was also observed in other studies even after formation of an authentic E2F1 complex derived from the adenoviral E2 gene promoter [38]. It is also possible that a different member of the E2F family binds at the PBK promoter [39,40]. Fig. 6. Codistribution of PBK and CREB factors by Western immunoblot analysis. Cell lysates from different tissues were tested for the presence of PBK and CREB factor using corresponding antibodies. Actin antibody was included as a loading control.
A double stranded oligomer overlapping to the E2F consensus binding site at position −146 bp in the PBK promoter was examined by the mobility shift assay using a Raji cell nuclear extract and HEK 293 nuclear extract (Fig. 7A and B). Both these cell lines express the PBK protein and should possess the relevant transcription factors in active form. The same specific gel shift complexes were observed in the Raji nuclear extract and the 293 nuclear extract, suggesting that
3.4. Major transcriptional activity overlaps with the minimal promoter retaining CREB and E2F binding sites In order to ascertain if E2F and CREB factors regulate PBK gene expression, we cloned the 2.5 kb promoter at BglII/NheI sites next to the promoterless red-emitting click beetle luciferase gene. The intact promoter fragment was subdivided into a proximal 0.6 kb fragment and a 2.0 kb distal Kpn fragment and luciferase activity was measured following transient transfection into 293 cells. A beta-galactosidase gene was cotransfected in order to normalize for transfection efficiency in those cells. These studies localized major pro-
Fig. 7. Gel mobility shift assay for E2F factor binding within the PBK promoter. A pair of complementary 39 mer oligos derived from the PBK promoter region covering the putative E2F binding site at −146 bp position were DIG-labeled and used in the gel shift assay utilizing Raji and HEK 293 nuclear extracts (A and B). The position of migration for the specific gel shift complex has been indicated. The asterisked band represents a nonspecific complex which was not competed by the unlabeled homologous competitor. E2FAb represents the E2F1 antibody.
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Fig. 8. Luciferase reporter assay to identify minimal PBK promoter. The putative PBK promoter spanning +27 to −2498 bp (with respect to the transcription start site) position was amplified and cloned next to the luciferase coding unit in the promoterless vector in the sense orientation. Luciferase activity derived from the 2.5 kb promoter was measured in parallel with the 0.6 kb (ATG-proximal) and 2.0 kb (ATG-distal) subfragments and the promoterless (Basic) vector following transfections into the 293 cells. A beta-galactosidase expression plasmid was cotransfected to normalize for possible variations in the transfection efficiency. Luciferase activities shown are derived from triplicate experiments.
moter activity for the PBK gene to within the ATG-proximal 0.6 kb fragment, which contains both E2F and CREB binding sites (Fig. 8). We also used the 2.5 kb luciferase construct to test if this promoter piece demonstrated suppressed activity in the presence of doxorubicin. Although doxorubicin mediated growth arrest could not be examined in HEK 293 cells since these cells lost adherence after prolonged incubation, we sought to examine the effect on the PBK promoter following transient transfection and subsequent incubation in the presence of 0.2 g/ml doxorubicin for a period of 48 h (Fig. 9). Luciferase expression from SV40 promoter was measured in order to
Fig. 9. Luciferase activity of reporter constructs under growth arrest by doxorubicin. The luciferase reporter construct with the 2.5 kb PBK promoter was transfected into 293 cells. Sixteen hours later, cells were treated with doxorubicin (0.2 g/ml) and were harvested at 48 h post-treatment for luciferase expression. An SV40 promoter driven luciferase construct was also transfected and treated under similar conditions as the PBK promoter construct. A beta-galactosidase expression plasmid was utilized as control in both transfection experiments. Luciferase activity derived from the 2.5 kb PBK promoter, untreated, which is 56.4% of the SV40 promoter activity has been shown as 100% for comparison of doxorubicin data.
Fig. 10. Western immunoblot analysis of knockdown extracts. The TranSilentTM Western blots (Panomics) containing extracts following siRNA inhibition of designated transcription factor expression in HEK293 cells were crossreacted with a monoclonal PBK antibody. Actin signals are shown to demonstrate equal protein loading. Selective depletion of transcription factors was demonstrated in the manufacturer’s data by crossreacting with IkB which shows absence of bands specifically in the p65 and p50 lanes (data not shown).
confirm that doxorubicin did not cause a generalized repression of other promoters as well. A beta-galactosidase plasmid was also included as a control for transfection efficiency. Downregulation of luciferase expression from the PBK promoter was observed in the presence of doxorubicin although the SV40 promoter function remained unaffected. Knockdown transcription factor expression was also utilized to determine if PBK expression in HEK 293 cells is dependent specifically upon E2F and CREB/ATF factors. Western blots containing equivalent amounts of knockdown extracts following siRNA inhibition of transcription factor expression were analyzed using monoclonal PBK antibody. As shown in Fig. 10, diminished expression of PBK was observed in extracts from HEK 293 cells which were engineered to downregulate expression of E2F1, CREB or ATF2 factor but not in the extracts derived from cells lacking NFkB (p65 and p50), Stat3 or SP3 factor. Taken together, these data indicate that the PBK promoter is subject to transcriptional downregulation during doxorubicin treatment and that the transcription factors E2F and CREB appear to be involved in this event. 3.5. Intact CREB/ATF and E2F binding sites are required for efficient transcription The effect of transcription factor binding at the promoter has been further examined by carrying out site-directed mutagenesis of the putative E2F and CREB/ATF binding sites and measuring promoter activity using the luciferase expression system. To this end the E2F and CREB/ATF binding sites have been sequentially mutated in the 0.6 kb proximal
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Fig. 11. Effect of abrogation of factor binding at the E2F or CREB/ATF sites in the PBK promoter. By site directed mutagenesis in the 0.6 kb luciferase promoter construct, factor binding at the E2F or CREB/ATF sites was abrogated. The E2F binding site was mutated by altering three nucleotides within the core binding motif while the CREB mutation involved a deletion in the core as described in the text. These altered promoters with single site mutations were tested by luciferase reporter assay and compared with the wild type 0.6 kb promoter construct. Luciferase activities shown, include the standard error means of four independent experiments.
promoter construct which retained approximately 95% of the total activity. The E2F core motif ‘GCGCCAAA’ was mutated to ‘GATACAAA’ based upon a similar strategy used to characterize the E2F binding site in the human c-myc gene promoter [41]. The ATF/CREB site was mutated by deleting four nucleotides in the core binding motif similar to the strategy used to characterize a CREB/ATF site in the human herpesvirus 6 DNA polymerase gene promoter [42]. The effect of mutating the E2F or CREB/ATF binding sites was tested in HEK 293 cells following transient transfection and measurement of luciferase activity. As shown in Fig. 11, abrogation of E2F or CREB binding caused reductions in the activity of the 0.6 kb promoter to 43 and 73% of the wildtype activity, respectively indicating that factor binding at these sites are critical for maintaining the proper level of PBK mRNA synthesis.
4. Discussion Cell cycle checkpoint activation leading to tumor cell senescence is being considered a potential target for novel therapeutic interventions [43–46]. p53 has an established role in the induction of G1 block and although there are G2 arrested cells, the major cell cycle arrest that takes place following DNA damage in the presence of genotoxic stress is the G1 block. In the presence of p53, G1 cell cycle arrest is mediated by p21waf1/cip1 which inhibits Cdk2 [47]. However, in the absence of functional p53, a frequent finding in advanced hematologic malignancies, cell cycle arrest takes place primarily at the G2 checkpoint. The mechanism leading to G2 block in the absence of p53 is an open question that is currently being addressed by several investigators. According to a recent report, p14ARF has been shown to be involved in
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negatively regulating Cdc2 activity by decreasing transcription as well as diminishing activity by preventing its nuclear localization [21]. Earlier it was reported that in human foreskin fibroblasts, loss of p53 function in the presence of E6 oncoprotein led to G2 arrest initiated by drugs such as doxorubicin which intercalate into DNA. The kinase activity of Cdc2 as well as its nuclear localization were not affected [48]. Depending upon the cell types involved in the process, there may be redundant pathways to cause G2 arrest that operate in the absence of p53. These pathways may or may not cause inactivation of Cdc2 kinase. Our studies show that promyelocytic leukemia (HL60) cells undergo G2 arrest in the presence of 0.2 g/ml doxorubicin. In addition, the level of PDZ-binding kinase (PBK) progressively decreases after cell cycle arrest. However, Cdc2 kinase remains dephosphorylated indicating that the mechanism of G2 arrest in HL60 cells is similar to that observed by Passalaris et al. [48]. Cell cycle arrest at G2/M has been confirmed by flow cytometry which shows enrichment of 4n cells following doxorubicin treatment. In this report we sought to examine the mechanism of PBK downregulation during doxorubicin induced cell cycle arrest. First, we found that the PBK mRNA level in HL 60 leukemic cells goes up as the cells are recruited into the G2/M phase followed by downregulation during sustained growth arrest. GAPDH mRNA which is constitutively expressed throughout the cell cycle, was maintained at a constant level. To further show that PBK mRNA undergoes cell cycle regulated expression, RNA analysis was carried out following synchronization of leukemia cells by the reversible DNA polymerase inhibitor, aphidicolin. These experiments demonstrated that PBK expression is cell cycle regulated in that PBK mRNA accumulates in the G1 and G2 phases of the cell cycle rather than in the S phase. In a recent report, Matsumoto et al. have shown that PBK protein expression is also cell cycle stage specific [9]. The promoter region for the PBK gene was analyzed to determine which putative transcription factors might be implicated in cell cycle regulated expression. These studies were performed in several different cell lines, which were known to express PBK at a high level, in order to establish a general mechanism for regulation. Sequence analysis revealed consensus E2F and CREB/ATF binding sites at −146 bp and −312 bp positions with respect to the transcription start site. To confirm an important role for E2F and CREB/ATF in transcriptional regulation, we have first shown binding of nuclear extract-transcription factors to the putative binding sites in the promoter by gel mobility shift assays in Raji and HeLa cell nuclear extracts. Second, by Western blot analysis we have shown that CREB factor is codistributed with the PBK protein in cells of variable tissue origin. Also, selective knockdown expression of CREB, ATF2 or E2F transcription factors, but not NFkB p65 and p50 subunits, SP3 and Stat3, was associated with diminished PBK expression in HEK 293 cells. By luciferase reporter assays, we have localized the major regulatory region for PBK expression to a 0.6 kb fragment encompassing the E2F
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and CREB binding sites. Finally, abrogation of factor binding at the E2F or the CREB site by site-directed mutagenesis, led to significant loss of PBK promoter function in HEK293 cell model. Thus, we favor a model that includes cooperation between E2F and CREB/ATF factors to optimally regulate PBK expression. A functional association between E2F and CREB factors has been previously predicted based upon an analysis of 124 genes whose promoters bind either to E2F1 or E2F4 in vivo [49]. Oncoprotein 18 (Op18) or stathmin is another cell cycle regulated gene. Stathmin is also downregulated during doxorubicin mediated growth arrest. It has been reported that loss of E2F1 binding and recruitment of E2F4, a transcriptional repressor to the same site provide a mechanism for downregulation during growth arrest employing a single site to both activate and repress transcription [22,50]. A similar mechanism may operate for PBK expression where E2F family of transcription factors play a major role in positive regulation during normal growth as well as negative regulation during growth arrest. In summary, our experiments demonstrate that PBK is regulated by cell cycle-specific transcription factors E2F and CREB/ATF. In addition, the expression from the PBK promoter is downregulated during doxorubicin-induced growth arrest. Loss of positive factor binding or binding of inhibitory factors belonging to these two important transcription factor families may explain the cell cycle-specific regulation which was observed and the negative regulation of PBK gene expression during growth arrest of leukemic cells. These studies provide insight into the regulation of a novel mitotic kinase which may be involved in leukemic cell growth and survival.
Acknowledgments We thank Dr. Paul Shapiro for intellectual guidance and for critical reading of the manuscript. We thank Katie Goetzinger, Jennie Hart, Jeffrey Kleinberg, Michael Landau and Dina Ioffe for laboratory assistance. This work was supported by an institutional grant (02-4-32303) from the University of Maryland School of Medicine and by unrestricted gifts from the Jiji Foundation and from Mr. Willard Hackerman. A.P.R is a Clinical Scholar of the Leukemia and Lymphoma Society.
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Expression of PDZ-binding kinase (PBK) is regulated by cell cycle-specific transcription factors E2F and CREB/ATF Asit K. Nandi ∗ , Aaron P. Rapoport University of Maryland School of Medicine, Greenebaum Cancer Center, 655 W Baltimore Street, Baltimore, MD 21201, USA Received 3 June 2005; received in revised form 9 August 2005; accepted 11 August 2005 Available online 19 September 2005
Abstract Earlier we reported that a novel mitotic protein kinase, PDZ-binding kinase (PBK), is expressed in primary hematopoietic neoplasms. Recent reports have suggested a role for PBK in mitotic progression. In the present study, we demonstrate that PBK is downregulated during doxorubicin induced growth arrest of HL60 promyelocytic leukemia cells at least partly due to cell cycle-specific transcriptional regulation. Furthermore, we show that transcriptional control is mostly due to binding of transcription factors E2F and CREB/ATF to two distinct binding sites within the PBK promoter. This was demonstrated by: (i) electrophoretic mobility shift assays showing transcription factor binding within the PBK promoter at the putative E2F (−146 bp) and CREB/ATF (−312 bp) binding sites; (ii) Western immunoblot analysis of knockdown extracts from siRNA inhibition of transcription factor expression showing that PBK protein expression is dependent upon the presence of these transcription factors; (iii) codistribution of CREB factor and PBK in cell lines of disparate tissue origin; and (iv) luciferase reporter assays showing that PBK promoter activity is dependent on factor binding at intact E2F and CREB/ATF sites. These findings may provide insight into the mechanisms that upregulate PBK expression in proliferative hematologic malignancies and downregulate its expression following growth arrest of leukemic cells. © 2005 Elsevier Ltd. All rights reserved. Keywords: PDZ-binding kinase; Cell cycle arrest; Transcription factors; E2F; CREB/ATF; Leukemic cells; PBK/TOPK
1. Introduction PDZ-binding kinase (PBK) or T cell originated protein kinase (TOPK) is a serine–threonine kinase, which is homologous to MAP kinase kinase MKK3 and has been reported to be activated during mitosis [1,2]. PBK was identified in our laboratory as an expressed sequenced tag (EST), which was differentially expressed in the Burkitt’s lymphoma cell line GA10 compared to hyperplastic tonsillar B cells [3]. PBK has been cloned as an interaction partner of the human tumor suppressor Discs-large (hdlg) by yeast two-hybrid screening of a HeLa cell cDNA library [1]. Other investigators cloned PBK as an IL-2 inducible gene in cytotoxic T lymphocytes and by protein–protein interactions with human C-Raf [2,4]. ∗
Corresponding author. Tel.: +1 410 328 1906; fax: +1 410 328 6559. E-mail addresses: [email protected] (A.K. Nandi), [email protected] (A.P. Rapoport). 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.08.011
The murine homologue was cloned as an IL-6 stimulated gene from myeloma cells [5]. PBK mRNA is expressed in a wide variety of tumor cell lines and in normal testicular and embryonic tissues [3]. Previous studies have shown that PBK protein is also expressed in primary clinical samples from patients with acute myelogenous leukemia (AML) and aggressive lymphoid neoplasms [6]. In contrast, PBK expression was barely detectable in CD34+ immunoselected peripheral blood progenitor cells. Consistent with previous findings, our data indicated that PBK phosphorylates p38 MAP kinase, thereby placing PBK as a MAP kinase kinase in the signal transduction pathway [7,8]. However, it is likely that other as yet unidentified cellular targets of PBK exist [9]. PDZ-binding kinase is activated possibly by Cdc2cyclinB during the mitotic phase [1]. Consequently, PBK may be a mitotic regulator. Similar to protein kinases PLK, Aurora kinases and NIMA, PBK may have a role in progres-
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sion through the mitotic phase [10–12]. In a recent report PBK/TOPK has been shown to be associated with mitotic spindles through a protein phosphorylation site recognized by Cdc2-cyclinB. Further studies involving knockdown RNA expression have suggested that PBK has a role in cytokinesis [9]. Studies using HL60 promyelocytic leukemia cells have shown strong downregulation of PBK protein expression after TPA-induced differentiation along the macrophage pathway [6,13,14]. Taken together, these data suggest that PBK expression may help regulate leukemic cell growth or survival. When exposed to genotoxic stress, many normal as well as tumor cells undergo cell cycle checkpoint activation and cell growth restriction in the G1 and G2 phases of the cell cycle [15–18]. Several regulators including p53 and p14ARF have a role in implementing the cell cycle blocks [19–21]. Downregulation of two mitotic regulators Stathmin and AIM1 following induction of G2/M arrest by the antineoplastic drug doxorubicin has been reported to be mediated by E2F factor [22–24]. A decrease in the transactivation function of the CREB factor also takes place in response to genotoxic stress [25–27]. Induction of cellular senescence by doxorubicin has been studied by several investigators [28–30]. Here we have examined the regulation of PBK during cellular senescence of HL60 promyelocytic leukemia cells following exposure to doxorubicin. We have found that PBK protein expression is downregulated during growth arrest by doxorubicin. We also demonstrate that the transcription factors E2F and CREB/ATF are critical regulators of PBK expression during growth arrest. These findings may have implications for the study of leukemic cell growth and survival.
2. Experimental procedures 2.1. Cell growth arrest and Western blot analysis Doxorubicin (Sigma) was dissolved in sterile water at a concentration of 2 mg/ml and filtered through 0.8/0.2 m syringe filters. The stock was diluted 1:10 (1000×) and stored at 4 ◦ C. HL60 cells were resuspended to a density of 4–6 × 105 cells/ml in the presence of 0.2 g/ml doxorubicin and maintained in the CO2 incubator at 37 ◦ C for varying periods of time. Aliquots of cells were taken out at specific time points, pelleted and resuspended in sterile PBS. Cells were lysed in 60 l CelLyticTM buffer (Sigma) containing protease inhibitors. The protein contents were measured using the BCA protein assay kit (Pierce) and aliquots containing 50 g of protein were run on 10% Tris-glycine gels and transferred to PVDF membranes according to the manufacturer’s protocol (Invitrogen). Membranes were blocked with ready to use blocking agent (KPL) for 1 h at 25 ◦ C and probed with a monoclonal antibody to PBK (BD Pharmingen) in the same blocking buffer for overnight at 4 ◦ C. The blots were processed according to standard methods and crossreacted with a HRP-linked secondary antibody (Amersham), washed
and developed using ECLTM detection reagents according to the supplier’s protocol (Amersham). Other antibodies utilized included actin antibody (Sigma), phospho-Cdc2 Tyr15 (cell signaling technology), and phospho-c-Myc antibodies (Cell Signaling Technology) all of which were blocked with 5% nonfat milk in TBST buffer. TranSilentTM Western blots containing lysates of human embryonic kidney 293 cells following knockdown of various transcription factors, were obtained from Panomics. Aphidicolin (Calbiochem) was dissolved at a concentration of 0.33 mg/ml in 70% ethanol and was used for S phase arrest at a final concentration of 3 g/ml for 24 h. Following this period, cells were washed in sterile PBS and suspended in prewarmed growth medium. 2.2. Genomic amplifications and construction of luciferase expression plasmids Genomic DNA was isolated from HL60 cells using High Pure PCR Template Preparation kit (Roche) and amplified using primers derived from the putative promoter of the PBK gene (5 -GCGCTAGCCTGAATTGCTGATGTTACAGG-3 , 5 -GCAGATCTCCTCTAGCACCAACACATACG-3 ) and a high fidelity DNA polymerase mix (Roche). BglII and NheI restriction sites were inserted in the primers. The cycling conditions were 95 ◦ C-1 , 51 ◦ C-3 , 68 ◦ C-2 for 35 cycles with an initial denaturation of 2.5 at 95 ◦ C and an extension period of 10 at 68 ◦ C at the end. The amplified 2.5 kb band, was gel purified and cloned into the PCRII TOPO T/A cloning vector (Invitrogen). The sequence of the amplified piece was confirmed following isolation of transformant clones derived from TOP10 cells. The BglII-NheI restriction fragment containing the 2.5 kb promoter in the sense orientation was cloned next to a luciferase coding sequence in the pCBRBasic vector (Promega). The 2.5 kb promoter construct in the pCBR-Basic vector was digested with KpnI to generate a 2.0 kb distal promoter fragment. The large Kpn fragment containing the rest of the plasmid including the 0.6 kb ATGproximal promoter piece next to the luciferase coding unit was also generated through religation. The 2.0 kb distal promoter was subsequently cloned next to the luciferase coding region in the sense orientation. Thus, two luciferase constructs were generated which contained the ATG-proximal promoter sequence from +27 to −558 bp position and the distal promoter sequences from −559 to −2498 bp position (with respect to the transcription start site). 2.3. RT-PCR In order to measure the level of PBK mRNA following doxorubicin treatment, total RNA was isolated at specific time points from 40 ml cultures using TrizoleTM reagent (Invitrogen). Aliquots equivalent to 2 g of total RNA were reverse transcribed and PCR amplified using onestep RT-PCR (Invitrogen) in the presence of two pairs of primers—one for amplification of the entire PBK coding
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region (5 -GCGGATCCCTTCCAGGCGGTGAGACTCTGGACT-3 and 5 -GCGTCGACGACATCTGTTTCCAGAGCTTCAAC-3 ) and the other for amplification of GAPDH as a control for constitutive gene expression (Q.Biogene). Conditions for thermal cycling were 50 ◦ C for 30 and 94 ◦ C for 2 followed by 30 cycles at 94 ◦ C-30 , 55 ◦ C-1 , 68 ◦ C-2’ and a final extension time of 7 at 72 ◦ C. 2.4. Electrophoretic mobility shift assay A pair of complementary 34-mers (5 -CCTATAATAAAATTGACGTCATAATCACGACACA-3 ) overlapping to the putative CREB-binding core motif ‘TGACGTCA’ present in the PBK promoter, were synthesized, annealed by slow cooling from 95 to 25 ◦ C and labeled using Digoxygenin11ddUTP following the manufacturer’s protocol (Roche). Gel shift assay for the CREB factor was carried out utilizing DIG-Gel Shift Kit (Roche) and 10 g of HeLa (Promega) or Raji (Active Motif) nuclear extracts. The complex was formed in the presence of poly[d(A–T)] as a nonspecific competitor and was resolved in precast 5% native polyacrylamide gel (Invitrogen) prerun for 1/2 h using 0.5 × TBE running buffer at 4 ◦ C. The E2F gel shift was carried out using a double stranded oligomer (5 -TTCCTTCCCTCGTCTTTGGCGCCTGCGGGCACCGGGAGT-3 ) and poly[d(IC)] as a nonspecific competitor in Raji and human embryonic kidney (HEK) 293 nuclear extracts (Active Motif). The gel run was carried out at 4 ◦ C for 2 h in 0.25× TBE buffer at 100 V. Following electrophoresis, the gels were blot transferred to positively charged nylon membranes utilizing the semidry blotting procedure, UV crosslinked and air dried. The membranes were subsequently developed following a chemiluminescent detection technique as specified by the manufacturer (Roche). To demonstrate the effect of antibodies towards the formation of specific gel shift complexes, nuclear extracts were preincubated with phospho-CREB (Ser133), phospho-ATF2 (Thr71) (Cell Signaling Technology) and E2F1 (Upstate) antibodies. 2.5. Luciferase assay HEK 293 cells (6–8 × 105 ) were plated in 60 mm tissue culture dishes 24 h before transfection. On the day of experiment, cells were fed with fresh medium 3–5 h prior to transfection. Two micrograms of the designated luciferase plasmid construct was mixed with 2.0 g of control betagalactosidase expression plasmid in a total volume of 25 l and were combined with 15 l FuGene6TM (Roche) and 100 l DME (no serum) according to protocol. The mixture was allowed to incubate for 15 min at room temp and was added to the cells without changing medium. At 48 h post-transfection, cells were harvested by scraping using sterile PBS and lysed in 250 l Glo-lysisTM buffer (Promega). The beta-galactosidase and luciferase assays were carried out using Beta-GloTM and Chroma-GloTM luciferase assay sys-
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tems (Promega) utilizing 2.5 l of 1:20 diluted extracts and 100 l of original extracts, respectively. Both luciferase and beta-galactosidase enzyme activities were measured in the TD 20/20 luminometer manually. 2.6. Site-directed mutagenesis The 0.6 kb proximal promoter construct was utilized to carry out site-directed mutagenesis of the putative E2F binding site following the quick change site-directed mutagenesis protocol (Stratagene). A pair of complimentary 33-mer oligonucleotides incorporating mutant bases (5 -CCTTCCCTCGTCTTTGTATCCTGCGGGCACCGG-3 ) were mixed with 10 ng of circular plasmid DNA in the appropriate buffer in the presence pfu ultra HF DNA polymerase and incubated in the thermal cycler programmed to undergo 20 cycles of 95 ◦ C-30 s, 55 ◦ C-1 min, 68 ◦ C-5 min following an initial denturation at 95 ◦ C for 30 s. The mixture of input and amplified DNA was digested directly with DpnI to eliminate parent DNA molecules and was transformed into XL-1 Blue cells. The mutant plasmids were indicated by loss of a NarI restriction cleavage site and further confirmed by sequencing using primers specific for the luciferase vector (Reporter Vector Primer3, Promega). Mutagenesis at the putative CREB binding site was carried out in the original PCRII TOPO vector with the entire 2.5 kb promoter fragment by deletion of four nucleotide 3 overhang ends following AatII digestion utilizing T4 DNA polymerase in the presence of dNTP [31]. The AatII restriction site was located within the core motif for CREB/ATF binding. Following confirmation by sequencing, the BglII/KpnI piece covering the 0.6 kb promoter along with the CREB site mutation was cloned into the pCBR-basic luciferase vector in the sense orientation. 2.7. Flow cytometry 1–2 × 106 Cells at specific time points were withdrawn from appropriately treated cultures, diluted with PBS (4 ◦ C) and were pelleted. Cells were resuspended thoroughly in 1 ml PBS, 2 ml 100% ethanol (−20 ◦ C) was added dropwise while shaking and the cell suspension was stored at −20 ◦ C until ready for flow cytometry. Two or three hours before analysis, cells were pelleted at 2000 rpm, suspended in 1 ml PBS, allowed to stand for 5 min, and were finally resuspended thoroughly in 1 ml propidium iodide-staining solution with RNase (BD Pharmingen) for 30 at 25 ◦ C. Cells were subsequently analyzed in FACScan (Becton Dickinson Instruments) utilizing the Cell Quest program. 3. Results 3.1. PBK protein is downregulated during growth arrest We first examined PBK expression in HL60 cells following treatment with doxorubicin. A variety of tumor cell
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Fig. 1. Doxorubicin induced cell cycle arrest of leukemic cells. The HL60 promyelocytic leukemia cells were treated with 0.2 g/ml doxorubicin (Dox), harvested after 0, 3, 6, 9, 12, 24 and 48 h as indicated and analyzed in the FACScan flow cytometer following RNase digestion and propidium iodide staining. M1, M2 and M3 represent peaks corresponding to the G1, S and G2/M phases, respectively. Only the G2% values are shown. The subdiploid population reflects less than 20% trypan blue positive cells, which did not change significantly during the course of the experiment.
lines, which do not carry a functional copy of the p53 gene undergo growth arrest in the G2/M phase in the presence of low doses of doxorubicin and the cells acquire senescencelike phenotype (SLP) [22,28–30]. Fig. 1 shows initiation of rapid cell growth arrest predominantly at the G2/M phase in the presence of 0.2 g/ml of doxorubicin. To further document growth arrest, the phosphorylation status of the Tyr 15 residue of the mitotic cyclin, cyclin B-regulated-cyclin dependent kinase (Cdc2) was examined. Phosphorylation of this site is carried out by Wee1 kinase which negatively regulates Cdc2 activity, while the Cdc25C phosphatase causes dephosphorylation of the Tyr15 residue. A decrease in the phosphorylated form of Cdc2 may indicate cell cycle arrest at the G2/M boundary [32–34] (see Section 4). As shown in Fig. 2, the level of Tyr 15 phosphorylation of Cdc2 decreased rapidly to an undetectable level by 48 h. While the HL 60 cells were growth-arrested, PBK expression diminished gradually at 48 and 72 h and at about 96 h post-treatment, PBK became nearly undetectable. The slow disappearance kinetics of PBK in this system is consistent with our previous observation regarding the rate of PBK disappearance after TPA induction of HL60 cells [6]. The level of phosphorylated (Ser780) retinoblastoma protein (Rb) remained constant for most of the time period and was slightly upregulated at 96 h and the level of phosphohistone-H3 (Ser10) remained constant (data not shown) indicating that there was no significant indiscriminate protein degradation. Actin levels also did not change during doxorubicin induction. In addition, cell viability was not affected significantly by doxorubicin treatment (80–90% viable cells by trypan blue exclusion).
Fig. 2. Downregulation of PBK protein level during growth arrest. Western immunoblot analysis of HL60 cell lysates after 0, 24, 48, 72 and 96 h of doxorubicin (0.2 g/ml) treatment. PBK was detected using a monoclonal PBK antibody. P-Cdc2 represents the levels of the Tyr15 phosphorylated form of Cdc2, a cyclin B dependent kinase at different timepoints. Phosphoc-Myc represents the level of the Thr58/Ser62 phosphorylated forms of cMyc protein. Stable actin and phospho-Rb levels are shown to demonstrate cell cycle arrest in the absence of generalized protein degradation. The minus (−) and plus (+) lanes represent control and apoptotic samples, respectively for Jurkat cells treated with camptothecin, to demonstrate that phospho-Rb disappears during apoptosis.
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We reported previously that phosphorylated c-Myc is downregulated during TPA induced differentiation of HL60 cells prior to PBK downregulation and that PBK was coexpressed with activated c-Myc in a number of human leukemias and lymphomas [6]. Therefore, c-Myc expression was monitored in the doxorubicin treated cells. Fig. 2 shows that downregulation of the phosphorylated form of c-Myc protein occurred prior to PBK downregulation. These results are in accordance with previous findings suggesting that PBK expression is correlated to active cellular growth and conditions which favor departure from the active growth phase either by inducing terminal differentiation or proliferation arrest, cause diminished expression of PBK. Thus, the c-myc gene being well known for its response to mitogenic or growth inhibitory stimuli, is co-regulated with PBK expression in these two models [35]. 3.2. Regulation of PBK involves cell cycle specific mRNA accumulation In order to examine whether PBK downregulation during growth arrest occurs at the transcriptional level or is caused by selective protein degradation, we analyzed PBK mRNA level by quantitative RT-PCR. Total RNA was isolated at different time points during doxorubicin treatment from HL60 cells, reverse transcribed and the cDNA was amplified in a single step in the presence of two sets of primers for PBK and GAPDH. The expected product for the GAPDH amplification was 516 bp while the other pair of primers amplified a 1 kb fragment covering the PBK coding region. As shown in Fig. 3A PBK mRNAs were maintained at a low level in asynchronous cells. After doxorubicin treatment, there was apparent upregulation possibly due to synchronization of cells in the G2 phase of the cell cycle within a period of 24 h followed by down regulation in the subsequent period as the cells become growth arrested. The 516 bp GAPDH product remained constant indicating that the amplification had been quantitative. These results indicate that PBK is negatively regulated at the level of mRNA following sustained cell cycle arrest of HL60 cells.
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PBK expression was further examined following synchronization of HL60 cells in the presence of the reversible DNA polymerase inhibitor aphidicolin and allowing resumption of cell cycle propagation. The level of PBK mRNA was significantly higher at the G1 phase (46.2% G1 at 0 h) compared to asynchronous cells and dropped when cells were predominantly in S phase (40.2% S at 4 h) (Fig. 3B). The level of PBK mRNA was again higher when cells were predominantly in the G2 phase (31.7% G2 at 8 h). At 12, 16 and 20 h the transcript levels maintained a similar profile. The levels of amplified GAPDH cDNA were nearly constant between 0 to 20 h. These results indicate that PBK mRNA accumulation is cell cycle dependent. These results in the HL60 cells are consistent with HeLa cell cycling studies involving microarray analysis, which show that PBK mRNA is cell cycle regulated [36]. In addition, a recent report by Matsumoto et al. also demonstrated that PBK/TOPK protein expression exhibits cell cycle specific regulation [9]. 3.3. PBK promoter encompasses binding sites for CREB/ATF and E2F transcription factors To study the mechanisms which regulate transcription, a 2.5 kb putative promoter for the PBK gene was amplified utilizing database sequence and genomic amplification. Human genomic DNA isolated from HL60 cells was amplified using primers designed to incorporate NheI and BglII restriction sites at the 5 and 3 ends of the promoter, respectively. On analysis, the PBK promoter did not contain a canonical ‘TATA’ box, but instead an A–T rich sequence occurred around −30 position with respect to the RNA start site [2,37]. Thus, PBK belongs to the class of ‘TATA’ less genes. We first determined if the PBK promoter region contained known transcription factor binding sites which could be implicated in cell cycle regulation. Based upon scanning for consensus transcription factor binding motifs, several putative transcription factor binding sites were localized within the 2.5 kb PBK promoter. We focused on two of the factor binding sites in the proximal part of the promoter. Thus, putative consensus binding sites were identified for E2F and CREB/ATF factors at positions −146 bp and −312 bp with respect to the RNA
Fig. 3. Cell cycle specificity of PBK mRNA accumulation by RT-PCR analysis. Total RNA was isolated from HL 60 cells following: (A) growth arrest by doxorubicin (0.2 g/ml) (DOX) and (B) cell synchronization in the presence of aphidicolin (3 g/ml) (APHI) for 24 h and subsequent growth in drug free medium for the designated periods. RNA analysis was carried out by one step RT-PCR and subsequent gel electrophoresis on agarose gels. The 1 kb amplification product was derived from PBK mRNA and the 516 bp product from GAPDH mRNA, respectively and have been identified. The PBK and GAPDH bands shown were derived from the same experiment.
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Fig. 4. Nucleotide sequence of a segment of the putative PBK promoter. The promoter sequences upstream of the PBK mRNA coding region has been derived from the Homo sapiens chromosome 8 genomic contig NT 007988 [gi:27478833]. Nucleotide position ‘+1 refers to the transcription start site corresponding to the 5 nucleotide of the PBK messenger RNA (1). The putative CREB/ATF and E2F binding sites have been shown. The underlined sequence represents an A/T rich cluster in place of a canonical TATA box.
start site for further study(Fig. 4). The selection of E2F and CREB/ATF was also motivated by the previous observation, which indicated that PBK was cell cycle regulated. Electrophoretic mobility shift assays were performed to determine if transcription factors in the nuclear extracts actually bind to the putative sites identified in the PBK promoter sequences, as stable DNA–protein interactions require further flanking sequences besides the core binding motifs. By electrophoretic mobility shift assay (EMSA) we detected fac-
tor binding at the CREB/ATF site using a HeLa cell nuclear extract (Fig. 5A) and also Raji nuclear extract (Fig. 5B). These cell lines were selected for analysis because they express the PBK protein at a high level. So, the key transcription factors, which regulate PBK gene expression should be present in these extracts. Binding was partially inhibited by both CREB antibody and ATF2 antibody but not by a heterologous E2F antibody indicating that CREB factor binds at the −312 bp position within the PBK promoter in the HeLa cell extract. Multiple complexes in the Raji nuclear extract possibly represent different combinations of CREB related factors that bind as homo-or heterodimers. A number of cell lines derived from multiple tissues of human, rat and mouse origin that were not previously analyzed were tested to determine if expression of transcription factor CREB coincides with PBK expression. Fig. 6 shows a Western blot analysis of CREB and PBK expression in the following cell lines: human fibroblasts (HS68), fibrosarcoma (HT1080), T cell leukemia (Jurkat) as well as mouse embryonic fibroblasts (C3H10T1/2), embryonal carcinoma (F9) and rat skeletal muscle myoblast (L6). Antibodies which crossreact with human, mouse and rat antigens were used and it was found that the two cell lines, Jurkat and F9 cells, which expressed PBK also expressed detectable levels of CREB factor. Notably, PBK was expressed at a low level in F9 cells which also expressed a low level of CREB factor, while the other lines had no detectable expression of either protein. Considered together these results suggested that CREB factor may regulate PBK expression in vivo.
Fig. 5. Gel mobility shift assay to demonstrate CREB/ATF factor binding within the PBK promoter at −312 bp position. A pair of double stranded 34 bp oligomers were derived from the PBK promoter encompassing the putative CREB binding site. These were labeled with Digoxygenin 11-ddUTP and were tested by the electrophoretic mobility shift assay using the HeLa nuclear extract (A) and Raji nuclear extract (B). Poly[d(A–T)] was used as a nonspecific competitor in this assay. A major band which is competed by the homologous unlabeled oligomers (“Homolog Comp”) has been identified as a specific gel shift complex. As expected a mutant competitor (“Mutant Comp”) showed no inhibitory activity in the Raji nuclear extract assay.
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E2F factor binds at the expected site within the PBK promoter. As shown in Fig. 7B, the faster migrating complex was not competed by unlabeled homologous competitor and was considered to be nonspecific. The slower migrating complex however demonstrated specific competition but was not affected by the addition of E2F1 antibody. Lack of interference by the presence of specific antibody was also observed in other studies even after formation of an authentic E2F1 complex derived from the adenoviral E2 gene promoter [38]. It is also possible that a different member of the E2F family binds at the PBK promoter [39,40]. Fig. 6. Codistribution of PBK and CREB factors by Western immunoblot analysis. Cell lysates from different tissues were tested for the presence of PBK and CREB factor using corresponding antibodies. Actin antibody was included as a loading control.
A double stranded oligomer overlapping to the E2F consensus binding site at position −146 bp in the PBK promoter was examined by the mobility shift assay using a Raji cell nuclear extract and HEK 293 nuclear extract (Fig. 7A and B). Both these cell lines express the PBK protein and should possess the relevant transcription factors in active form. The same specific gel shift complexes were observed in the Raji nuclear extract and the 293 nuclear extract, suggesting that
3.4. Major transcriptional activity overlaps with the minimal promoter retaining CREB and E2F binding sites In order to ascertain if E2F and CREB factors regulate PBK gene expression, we cloned the 2.5 kb promoter at BglII/NheI sites next to the promoterless red-emitting click beetle luciferase gene. The intact promoter fragment was subdivided into a proximal 0.6 kb fragment and a 2.0 kb distal Kpn fragment and luciferase activity was measured following transient transfection into 293 cells. A beta-galactosidase gene was cotransfected in order to normalize for transfection efficiency in those cells. These studies localized major pro-
Fig. 7. Gel mobility shift assay for E2F factor binding within the PBK promoter. A pair of complementary 39 mer oligos derived from the PBK promoter region covering the putative E2F binding site at −146 bp position were DIG-labeled and used in the gel shift assay utilizing Raji and HEK 293 nuclear extracts (A and B). The position of migration for the specific gel shift complex has been indicated. The asterisked band represents a nonspecific complex which was not competed by the unlabeled homologous competitor. E2FAb represents the E2F1 antibody.
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Fig. 8. Luciferase reporter assay to identify minimal PBK promoter. The putative PBK promoter spanning +27 to −2498 bp (with respect to the transcription start site) position was amplified and cloned next to the luciferase coding unit in the promoterless vector in the sense orientation. Luciferase activity derived from the 2.5 kb promoter was measured in parallel with the 0.6 kb (ATG-proximal) and 2.0 kb (ATG-distal) subfragments and the promoterless (Basic) vector following transfections into the 293 cells. A beta-galactosidase expression plasmid was cotransfected to normalize for possible variations in the transfection efficiency. Luciferase activities shown are derived from triplicate experiments.
moter activity for the PBK gene to within the ATG-proximal 0.6 kb fragment, which contains both E2F and CREB binding sites (Fig. 8). We also used the 2.5 kb luciferase construct to test if this promoter piece demonstrated suppressed activity in the presence of doxorubicin. Although doxorubicin mediated growth arrest could not be examined in HEK 293 cells since these cells lost adherence after prolonged incubation, we sought to examine the effect on the PBK promoter following transient transfection and subsequent incubation in the presence of 0.2 g/ml doxorubicin for a period of 48 h (Fig. 9). Luciferase expression from SV40 promoter was measured in order to
Fig. 9. Luciferase activity of reporter constructs under growth arrest by doxorubicin. The luciferase reporter construct with the 2.5 kb PBK promoter was transfected into 293 cells. Sixteen hours later, cells were treated with doxorubicin (0.2 g/ml) and were harvested at 48 h post-treatment for luciferase expression. An SV40 promoter driven luciferase construct was also transfected and treated under similar conditions as the PBK promoter construct. A beta-galactosidase expression plasmid was utilized as control in both transfection experiments. Luciferase activity derived from the 2.5 kb PBK promoter, untreated, which is 56.4% of the SV40 promoter activity has been shown as 100% for comparison of doxorubicin data.
Fig. 10. Western immunoblot analysis of knockdown extracts. The TranSilentTM Western blots (Panomics) containing extracts following siRNA inhibition of designated transcription factor expression in HEK293 cells were crossreacted with a monoclonal PBK antibody. Actin signals are shown to demonstrate equal protein loading. Selective depletion of transcription factors was demonstrated in the manufacturer’s data by crossreacting with IkB which shows absence of bands specifically in the p65 and p50 lanes (data not shown).
confirm that doxorubicin did not cause a generalized repression of other promoters as well. A beta-galactosidase plasmid was also included as a control for transfection efficiency. Downregulation of luciferase expression from the PBK promoter was observed in the presence of doxorubicin although the SV40 promoter function remained unaffected. Knockdown transcription factor expression was also utilized to determine if PBK expression in HEK 293 cells is dependent specifically upon E2F and CREB/ATF factors. Western blots containing equivalent amounts of knockdown extracts following siRNA inhibition of transcription factor expression were analyzed using monoclonal PBK antibody. As shown in Fig. 10, diminished expression of PBK was observed in extracts from HEK 293 cells which were engineered to downregulate expression of E2F1, CREB or ATF2 factor but not in the extracts derived from cells lacking NFkB (p65 and p50), Stat3 or SP3 factor. Taken together, these data indicate that the PBK promoter is subject to transcriptional downregulation during doxorubicin treatment and that the transcription factors E2F and CREB appear to be involved in this event. 3.5. Intact CREB/ATF and E2F binding sites are required for efficient transcription The effect of transcription factor binding at the promoter has been further examined by carrying out site-directed mutagenesis of the putative E2F and CREB/ATF binding sites and measuring promoter activity using the luciferase expression system. To this end the E2F and CREB/ATF binding sites have been sequentially mutated in the 0.6 kb proximal
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Fig. 11. Effect of abrogation of factor binding at the E2F or CREB/ATF sites in the PBK promoter. By site directed mutagenesis in the 0.6 kb luciferase promoter construct, factor binding at the E2F or CREB/ATF sites was abrogated. The E2F binding site was mutated by altering three nucleotides within the core binding motif while the CREB mutation involved a deletion in the core as described in the text. These altered promoters with single site mutations were tested by luciferase reporter assay and compared with the wild type 0.6 kb promoter construct. Luciferase activities shown, include the standard error means of four independent experiments.
promoter construct which retained approximately 95% of the total activity. The E2F core motif ‘GCGCCAAA’ was mutated to ‘GATACAAA’ based upon a similar strategy used to characterize the E2F binding site in the human c-myc gene promoter [41]. The ATF/CREB site was mutated by deleting four nucleotides in the core binding motif similar to the strategy used to characterize a CREB/ATF site in the human herpesvirus 6 DNA polymerase gene promoter [42]. The effect of mutating the E2F or CREB/ATF binding sites was tested in HEK 293 cells following transient transfection and measurement of luciferase activity. As shown in Fig. 11, abrogation of E2F or CREB binding caused reductions in the activity of the 0.6 kb promoter to 43 and 73% of the wildtype activity, respectively indicating that factor binding at these sites are critical for maintaining the proper level of PBK mRNA synthesis.
4. Discussion Cell cycle checkpoint activation leading to tumor cell senescence is being considered a potential target for novel therapeutic interventions [43–46]. p53 has an established role in the induction of G1 block and although there are G2 arrested cells, the major cell cycle arrest that takes place following DNA damage in the presence of genotoxic stress is the G1 block. In the presence of p53, G1 cell cycle arrest is mediated by p21waf1/cip1 which inhibits Cdk2 [47]. However, in the absence of functional p53, a frequent finding in advanced hematologic malignancies, cell cycle arrest takes place primarily at the G2 checkpoint. The mechanism leading to G2 block in the absence of p53 is an open question that is currently being addressed by several investigators. According to a recent report, p14ARF has been shown to be involved in
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negatively regulating Cdc2 activity by decreasing transcription as well as diminishing activity by preventing its nuclear localization [21]. Earlier it was reported that in human foreskin fibroblasts, loss of p53 function in the presence of E6 oncoprotein led to G2 arrest initiated by drugs such as doxorubicin which intercalate into DNA. The kinase activity of Cdc2 as well as its nuclear localization were not affected [48]. Depending upon the cell types involved in the process, there may be redundant pathways to cause G2 arrest that operate in the absence of p53. These pathways may or may not cause inactivation of Cdc2 kinase. Our studies show that promyelocytic leukemia (HL60) cells undergo G2 arrest in the presence of 0.2 g/ml doxorubicin. In addition, the level of PDZ-binding kinase (PBK) progressively decreases after cell cycle arrest. However, Cdc2 kinase remains dephosphorylated indicating that the mechanism of G2 arrest in HL60 cells is similar to that observed by Passalaris et al. [48]. Cell cycle arrest at G2/M has been confirmed by flow cytometry which shows enrichment of 4n cells following doxorubicin treatment. In this report we sought to examine the mechanism of PBK downregulation during doxorubicin induced cell cycle arrest. First, we found that the PBK mRNA level in HL 60 leukemic cells goes up as the cells are recruited into the G2/M phase followed by downregulation during sustained growth arrest. GAPDH mRNA which is constitutively expressed throughout the cell cycle, was maintained at a constant level. To further show that PBK mRNA undergoes cell cycle regulated expression, RNA analysis was carried out following synchronization of leukemia cells by the reversible DNA polymerase inhibitor, aphidicolin. These experiments demonstrated that PBK expression is cell cycle regulated in that PBK mRNA accumulates in the G1 and G2 phases of the cell cycle rather than in the S phase. In a recent report, Matsumoto et al. have shown that PBK protein expression is also cell cycle stage specific [9]. The promoter region for the PBK gene was analyzed to determine which putative transcription factors might be implicated in cell cycle regulated expression. These studies were performed in several different cell lines, which were known to express PBK at a high level, in order to establish a general mechanism for regulation. Sequence analysis revealed consensus E2F and CREB/ATF binding sites at −146 bp and −312 bp positions with respect to the transcription start site. To confirm an important role for E2F and CREB/ATF in transcriptional regulation, we have first shown binding of nuclear extract-transcription factors to the putative binding sites in the promoter by gel mobility shift assays in Raji and HeLa cell nuclear extracts. Second, by Western blot analysis we have shown that CREB factor is codistributed with the PBK protein in cells of variable tissue origin. Also, selective knockdown expression of CREB, ATF2 or E2F transcription factors, but not NFkB p65 and p50 subunits, SP3 and Stat3, was associated with diminished PBK expression in HEK 293 cells. By luciferase reporter assays, we have localized the major regulatory region for PBK expression to a 0.6 kb fragment encompassing the E2F
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and CREB binding sites. Finally, abrogation of factor binding at the E2F or the CREB site by site-directed mutagenesis, led to significant loss of PBK promoter function in HEK293 cell model. Thus, we favor a model that includes cooperation between E2F and CREB/ATF factors to optimally regulate PBK expression. A functional association between E2F and CREB factors has been previously predicted based upon an analysis of 124 genes whose promoters bind either to E2F1 or E2F4 in vivo [49]. Oncoprotein 18 (Op18) or stathmin is another cell cycle regulated gene. Stathmin is also downregulated during doxorubicin mediated growth arrest. It has been reported that loss of E2F1 binding and recruitment of E2F4, a transcriptional repressor to the same site provide a mechanism for downregulation during growth arrest employing a single site to both activate and repress transcription [22,50]. A similar mechanism may operate for PBK expression where E2F family of transcription factors play a major role in positive regulation during normal growth as well as negative regulation during growth arrest. In summary, our experiments demonstrate that PBK is regulated by cell cycle-specific transcription factors E2F and CREB/ATF. In addition, the expression from the PBK promoter is downregulated during doxorubicin-induced growth arrest. Loss of positive factor binding or binding of inhibitory factors belonging to these two important transcription factor families may explain the cell cycle-specific regulation which was observed and the negative regulation of PBK gene expression during growth arrest of leukemic cells. These studies provide insight into the regulation of a novel mitotic kinase which may be involved in leukemic cell growth and survival.
Acknowledgments We thank Dr. Paul Shapiro for intellectual guidance and for critical reading of the manuscript. We thank Katie Goetzinger, Jennie Hart, Jeffrey Kleinberg, Michael Landau and Dina Ioffe for laboratory assistance. This work was supported by an institutional grant (02-4-32303) from the University of Maryland School of Medicine and by unrestricted gifts from the Jiji Foundation and from Mr. Willard Hackerman. A.P.R is a Clinical Scholar of the Leukemia and Lymphoma Society.
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