Coexpression and regulation of the FGF-2 and FGF antisense genes in leukemic cells

Leukemia Research 29 (2005) 423–433

Coexpression and regulation of the FGF-2 and FGF antisense genes in leukemic cells Mark Baguma-Nibashekaa,1 , Audrey W. Lia,1 , Mohammed S. Osmanb , Laurette Geldenhuysc , Alan G. Cassonc,d , Catherine K.L. Toob,e , Paul R. Murphya,e,∗ b

a Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5 c Department of Pathology, Faculty of Medicine, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5 d Department of Surgery, Faculty of Medicine, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5 e Department of Obstetrics and Gynaecology, Faculty of Medicine, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5

Received 31 May 2004; accepted 14 September 2004 Available online 30 December 2004

Abstract Fibroblast growth factor-2 (FGF-2) is a growth and survival factor whose expression is elevated in many hematopoietic malignancies. A natural antisense RNA (FGF-AS) has been implicated in the posttranscriptional regulation of FGF-2 mRNA expression. We demonstrate for the first time that FGF sense and antisense RNAs are coordinately expressed and translated in hematopoietic cells and tissues. Cytokine stimulation of growth-arrested K562 cells elicited a rapid transient increase in FGF-AS mRNA expression followed by a slower but sustained increase in FGF-2 mRNA. This was accompanied by a marked increase in the expression and nuclear translocation of FGF-2 and the FGF-AS encoded protein, GFG/NUDT6. These findings suggest a role for both FGF-2 and GFG proteins in the cell survival and proliferation of lymphoid and myeloid tumor cells. © 2004 Elsevier Ltd. All rights reserved. Keywords: Fibroblast growth factor; FGF-2; Antisense; Lymphoma; Nuclear

1. Introduction Basic fibroblast growth factor (FGF-2) is the prototypic member of a burgeoning family of related genes encoding heparin-binding proteins with growth-, anti-apoptotic- and differentiation-promoting activity [1]. Several lines of evidence have implicated FGF-2 in the development and progression of hematological tumors (reviewed in [2]). Increased levels of FGF-2 immunoreactivity are detected in patients with a variety of hematopoietic diseases including chronic myeloid and lymphoid leukemias [3], hairy cell leukemia [4], and non-Hodgkin’s lymphoma [5]. Furthermore, elevated ∗ 1

Corresponding author. Tel.: +1 902 4941579; fax: +1 902 4942050. E-mail address: [email protected] (P.R. Murphy). These authors contributed equally to the work.

0145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2004.09.006

intracellular levels of FGF-2-like immunoreactivity in lymphocytes are correlated with a high-risk stage of chronic lymphocytic leukemia (CLL), and with reduced susceptibility to apoptosis [6]. FGF-2 may protect against apoptosis in lymphocytes from patients with CLL and in CLL leukemic cell lines [6,7]. In addition, FGF-2 release by leukemic cells may support tumor progression by direct stimulation of bone marrow angiogenesis [5]. These findings suggest that abnormal regulation of FGF-2 expression may be a common event in the development of hematopoietic malignancies [8]. Although the control of FGF-2 expression is poorly understood, one intriguing possibility is regulation by a natural antisense RNA, FGF-AS [9]. FGF-2 and FGF-AS are transcribed as 3 to 3 overlaps (i.e., tail to tail overlapping in their 3 ends (Fig. 1) and have been shown to form stable double-stranded RNA duplexes in vivo [10]. The organization

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Fig. 1. Bi-directional transcription and alternative splicing of the FGF/FGF-AS genes. (A) Bi-directional transcription generates FGF-2 and FGF-AS transcripts which overlap at their 3 ends. Xenopus, rat and human FGF-AS transcripts are shown here. Regions of complementarity of FGF-AS with the human FGF-2 mRNA are indicated by the dashed lines. The locations of RT-PCR primers used for amplification of FGF-2 and alternatively spliced human and rat FGF-AS mRNAs are indicated by the small arrows. See Table 1 for details. (B) RT-PCR detection of FGF-AS mRNA splice variants. Left panel: exon-specific primers listed in Table 1 were used to selectively amplify the FGF-AS 1A and 1B isoforms in human liver RNA. Total FGF-AS (C) was detected using a primer set which amplified a portion of exons 2 and 3 common to both FGF-AS mRNAs. Right panel: rat Nb2 lymphoma RNA, and control cDNAs encoding the full length FGF-AS (FL), or alternatively spliced variants 2 or 2/3, were amplified using primers spanning the alternatively spliced exons. Nb2 cells expressed only the full length FGF-AS mRNA.

and sequence of the FGF-2 and FGF-AS transcripts have been highly conserved from amphibian to human, suggesting a functional importance for this structural relationship (reviewed in [11]). FGF-AS encodes the protein GFG/NUDT6, a member of the nudix family of nucleoside phosphohydrolases, whose function is currently unknown [12,13]. The hypothesis that FGF-2 may be regulated by interaction with the FGF antisense RNA is supported by the inverse association of FGF-2 and FGF-AS mRNA abundance seen in a variety of tissues across several species [14–20]. We recently demonstrated that forced overexpression of the FGF-AS mRNA effectively suppressed FGF-2 levels in stably transfected cells [9]. There are conflicting reports regarding FGF gene expression in leukemic cell lines. Krejci et al. [8] reported that FGF2 was expressed by the majority of leukemia and lymphoma cell lines examined. In contrast, Gagnon et al. [19] reported that FGF-AS was the only FGF-related mRNA expressed, and that FGF-2 was undetectable. The expression of the FGF-AS

encoded protein in human tissues has not been reported. The primary purpose of this study was to examine the expression of FGF-2 and FGF-AS mRNAs and their cognate proteins in rat and human lymphoid cell lines and tissues.

2. Materials and methods 2.1. Growth factors and antibodies Human prolactin (PRL) was a generous gift of Dr. Robert Shiu (Department of Physiology, University of Manitoba, Canada). Recombinant interleukin-2 (IL-2) and FGF-2 (both human) were obtained from Upstate Biotechnology (Lake Placid, NY). A polyclonal antiserum raised against bovine pituitary FGF-2 was generously provided by Dr. Claudia Goethe (Department of Anatomy and Cell Biology, Philipps-University of Marburg, Germany). Affinity-purified anti-FGF-2 (Ab-2) polyclonal antibodies raised against a

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synthetic peptide corresponding to amino acids 40–63 of human FGF-2 was obtained from Oncogene Research Products (Cambridge, MA). The polyclonal antibody against the carboxy-terminal domain of the antisense FGF protein (GFG) has been previously characterized [16]. Donkey antirabbit IgG-horseradish peroxidase conjugate was obtained from Amersham Pharmacia Biotechnology (Baie d’Urfe, PQ, Canada). AlexaFluor antibodies were purchased from Molecular Probes Inc. (Eugene, OR). 2.2. Cell culture Suspension cultures of the rat PRL-dependent Nb2-11C (Nb2) T-lymphoma cell line were maintained in Fischer’s medium for leukemic cells containing 10% fetal bovine serum (FBS) as a source of lactogens and 10% lactogenfree horse serum (HS) as previously described [21,22]. Nb2 cells express receptors for IL-2, which also acts as a mitogen in these cells. The HS used in these studies did not support growth of Nb2 cells indicating that it did not contain IL-2. Nb2 cells were growth-arrested at a cell density of ∼1.0 × 106 cells ml−1 in medium containing 10% HS alone for 18–24 h. For expression studies, quiescent cells at 0.2 × 106 cells ml−1 in 10% HS-medium were treated with PRL (10 ng ml−1 ) or IL-2 (12 U ml−1 ) for the indicated times whereas controls were untreated. Human cell lines were obtained from ATCC and maintained as recommended by the supplier. For investigation of IL-2 inducible gene expression, K562 human myeloid leukemic cells were cultured for 24 h at a density of 1 × 106 cells ml−1 in RPMI medium containing 5% IL-2 free horse serum (HS). After 24 h the medium was replaced with the same volume of fresh 5% HS-RPMI prior to IL-2 addition. For investigation of serum-inducible gene expression, K562 cells were growth restricted in 0.5% FBS/RPMI for 24 h at a density of 1 × 106 cells ml−1 . At 0 h, FBS was added to the cultures to a final concentration of 10%. 2.3. RT-PCR DNA-free total RNAs were extracted and amplified by RT-PCR as previously described [9]. Primary human tissue

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samples were from the BD Multiple tissue cDNA Panel (BD Biosciences, Mississauga, ON). The locations of the primers for FGF-2 and FGF-AS amplification are shown in Fig. 1. Primer sequences and expected product sizes are detailed in Table 1. The FGF-2 and FGF-AS mRNA-specific primer pairs F1/F2, and P1/P2 amplify products of 352 and 301 bp, respectively, from both human and rat RNA [16,23]. The full length 1060 bp rat FGF-AS mRNA was amplified using the GFG5/R3A primer pair as previously described in [9]. A 198 bp region of the FGF-AS mRNA common to both A and B splice variants was amplified using primers C1 and C2, previously described by Gagnon et al. [19]. The human FGFAS 1A and 1B isoforms were amplified using exon-specific forward and reverse primers to generate products of 323 and 217 bp, respectively. The two sets of 18S primers used, giving TM either 315 or 488 bp products were from the QuantumRNA 18S Internal Standards Kit (Ambion Inc.). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) primers were provided with the BD Multiple Tissue cDNA Panel kit. All PCR products were obtained within the linear range of the reaction. 2.4. Extraction and Western analysis FGF-2 and GFG FGF-2 extraction was performed as described [24] with some modification. Briefly, cells were washed and homogenized in lysis buffer (500 ␮l of 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA and 50 mM sodium pyrophosphate, containing 3 ␮g ml−1 each of aprotinin, leupeptin, pepstatin and 2 mM phenylmethylsulfonyl fluoride). After 30 min on ice, the homogenates were passed gently through 21 g needles. Cell nuclei were obtained by centrifugation (800 × g, 10 min), washed once with 250 ␮l of lysis buffer and repelleted. The 800 × g supernatants (cytoplasm) were pooled and the NaCl concentration adjusted to 0.5 mM. Nuclear pellets were resuspended in 500 ␮l of lysis buffer but containing 1 M NaCl to extract FGF-2. After centrifugation at 25,000 × g for 30 min, the supernatants were collected and adjusted to 0.5 M NaCl by addition of 10 mM Tris–1 mM EDTA containing protease inhibitors. Crude nuclear extracts and cytoplasmic fractions were adsorbed to heparin-Sepharose (Pharmacia Fine Chemicals, Dorval,

Table 1 Primers used for RT-PCR amplification of human and rat FGF and FGF-AS Target

Species/isoform

Forward primer

FGF-2

Human/rat

F1

FGF-AS

Human/rat

P1

FGF-AS

Rat (full)

GFG5

FGF-AS

Human

C1

FGF-AS

Human 1A

A1

FGF-AS

Human 1B

B1

5 -GGCTTCTTCCTGCGCATCCA-3 5 -ATGTGGAAGTTTCCAGGAGGCCTGTCA-3 5 -CAAGGTGCAACAATGTGGTGGGCGAG-3 5 -CTGCAGTACAGCAATGGCGA-3 5 -ACAGCTGTAACGGCATCTGTGAAAG-3 5 -CGCTGGCGCGCGATGCTTGCCCGAAC-3

Reverse primer F2 P2 R3A C2 A2 B2

5 -GCTCTTAGCAGACATTGGAAGA-3 5 -GCCTAGCAACTCTGCTGGTGATGGGAG-3 5 -GTCACGTATTTCCTTTATTTGAAAAGAGG-3 5 -CCTACTTGATGTGAAGCATATCCTG-3 5 -CTGCTGGGCCCTTCTCTCAGCCACAGAG-3 5 -GCCTGCAAGCCCTTCTGGAAGGCGG-3

Product (bp) 352 301 1060 198 323 217

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PQ) overnight at 4 ◦ C with constant mixing. The heparinSepharose pellets (25 ␮l packed bed volume) were washed with Tris–EDTA containing 0.5 M NaCl and the heparinbound proteins were eluted by boiling in Laemmli loading buffer. For analysis of GFG, total cell lysates were prepared in ice-cold lysis buffer. Proteins (10–20 ␮g/lane) were electrophoresed on 4–20% linear gradient or 12% SDS-PAGE gels for Western blot analyses. Immunoreactive signals were detected with Super Signal ULTRA (Pierce, Rockford, IL). 2.5. Immunohistochemistry and immunofluorescent confocal microscopy An indirect immunoperoxidase assay was used to evaluate cellular protein distribution of FGF-2 and GFG in formalin-fixed, paraffin-embedded human tissue sections (Imgenex Histoarray, BIO/CAN Scientific, Mississauga, ON), using primary antibodies at 2 ␮g ml−1 (anti-FGF-2) and 20 ␮g ml−1 (anti-GFG) dilution. Controls were run in parallel with test sections, and included known positive and negative staining tissues. All assays were repeated in duplicate. Tissue sections were evaluated independently by two investigators, and consensus reached at a double-headed microscope. To overcome potential limitations of subjectivity, variable immunoreactivity and tissue heterogeneity, a score comprising both the fraction of immunopositive cells (proportion score: 0: none; 1: less than one-hundredth; 2: one-hundredth to onetenth; 3: one-tenth to one-third; 4: one-third to two-thirds; 5: greater than two-thirds) and intensity of staining (intensity score: 0: none; +: weak; ++: intermediate; +++: strong) was assigned to each tissue section, noting subcellular protein distribution (nuclear or cytoplasmic). A composite score was used to signify accumulation of FGF-2 and GFG protein in each tissue section. Cells were prepared for immunofluorescence studies as previously described [25]. Briefly, following appropriate incubation, cells were washed twice with cold phosphate buffered saline (PBS). Cells were cytospun at 90 × g for 7 min onto each silanized microscope slide, fixed with −20 ◦ C acetone for 2 min, air-dried and kept at −70 ◦ C until further processing. Slides were treated with 1% paraformaldehyde in 60 mM l-lysine–0.1 M disodium hydrogen orthophosphate, pH 7.4 for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min and then blocked with 3% BSA in PBS for 1 h. The slides were exposed sequentially to the primary antibody (anti-FGF-2; 10 ␮g ml−1 in 0.1% BSA-PBS or antiGFG 350 ␮g ml−1 ) and a fluorescent tagged secondary antibody (AlexaFluorTM 488 [26] goat anti-rabbit IgG conjugate; 40 ␮g ml−1 in 0.1% BSA-PBS), each for 1 h at room temperature, followed by DNA staining using propidium iodide (0.05 ␮g ml−1 in 0.1% BSA-PBS) for 10 min. Slides were mounted in a drop of glycerol-PBS (CitifluorTM , Marivac, Halifax, NS, Canada). Controls included application of the secondary antibody without prior exposure of the cells to the primary, or following preabsorption of the primary antibody with the immunizing peptide. Image analysis used the stan-

dard operating software on the Zeiss LSM 510 microscope, and images requiring the comparison of fluorescence intensities following different periods of experimental treatment were taken at identical settings of laser intensity, pinhole, gain and black level. 2.6. Statistical analysis ANOVA and Scheffe’s F-test were performed using Statview (Abacus Concepts Inc., Berkeley, CA). Differences of p < 0.05 were taken as significant.

3. Results 3.1. Rat and human leukemic cells co-express FGF-2 and FGF-AS mRNAs As the FGF-AS gene is subject to a process of alternative splicing in both humans and rats, we employed RT-PCR using species- and exon-specific primers to distinguish FGF-AS alternative splice variants in both human and rat tissues. The positions of alternative splice-specific primers are shown in Fig. 1A, and their sequences in Table 1. Human FGF-AS A and B splice forms were expressed in approximately equal abundance in human liver, a tissue we have previously shown to be a major site of FGF-AS expression (Fig. 1B, left panel). Rat Nb2 lymphoma cells exclusively expressed the full length FGF-AS mRNA (Fig. 1B, right panel). We next examined the expression of FGF-2 and FGF-AS in human lymphoid tissues and cell lines. As shown in Fig. 2, FGF-AS was the major FGF RNA species expressed in human thymus, spleen and peripheral blood leukocytes (PBL). FGF-2 mRNA was detectable in thymus, but undetectable in normal spleen or PBL. In contrast, FGF-2 and FGF-AS were strongly co-expressed in human placenta and colon. Human cell lines examined included Jurkat (T cell leukemia), K562 (chronic myeloid leukemia), CCRF-CEM (acute lymphoblastic leukemia), U937 (histiocytic lymphoma), HMC-1 (mast cell leukemia) and its subclone HMC-5c6 In the majority of human leukemic cell lines examined, including Jurkat, K562 and CCRF-CEM cells, FGF-AS was the major FGF mRNA expressed, whereas FGF-2 was at the limit of detection in all lines except the U937 (promonocytic) and the HMC-5C6 mast cell line (Fig. 2B). Although total FGF-AS expression (FGF-AS C) was approximately equal in all lines examined, the alternative splice variant 1A was the major isoform in Jurkat, K562, U937 and HMC-1 cells. FGF-AS 1B was the major FGF-AS isoform expressed in CCRF-CEM and HMC-5C6 cells. Western blot analysis of human and rat leukemic cell lysates with an antiserum against the C-terminus of the rat GFG sequence demonstrated the presence of a 35 kDa translation product, which is predicted from the open reading frame of the FGF-AS mRNA (Fig. 2C). Rat Nb2 cells expressed the 35 kDa isoform exclusively whereas the human

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tyes in a histologically normal lymph node (Fig. 3, panels b and d). FGF-2 was not expressed in normal lymphocytes (Fig. 3b), but was detected in the cytoplasm of immunoblastic lymphoma cells (Fig. 3a). By contrast, cytoplasmic and nuclear immunoreactivity for GFG was seen in immunoblastic lymphoma cells (Fig. 3, panel c). In normal lymphocytes, only cell nuclear GFG immunoreactivity was seen (Fig. 3, panel d). 3.2. Regulation of FGF-2 and FGF-AS expression by growth arrest and mitogen stimulation

Fig. 2. Detection of human FGF-AS mRNA and protein in human tissues and cell lines. (A) Human PCR-ready tissue cDNAs were amplified using specific primers for FGF-2, FGF-AS and FGF-AS 1A and 1B splice variants. Approximately equal RNA input was confirmed by amplification of GAPDH as a loading control. The lanes are 1, placenta; 2, negative control (no cDNA); 3, spleen; 4, thymus; 5, peripheral blood leukocytes; and 6, colon. (B) RTPCR amplification of FGF-2 and FGF-AS in hematopoietic cell lines. Lanes: 1, Jurkat T cell leukemia; 2, K562 chronic myeloid leukemia; 3, CCRFCEM acute lymphoblastic leukemia; U937 histiocytic lymphoma; 5, HMC1 mast cell leukemia; 6, HMC-5C-6 mast cell leukemia. (C) Western blot detection of the FGF-AS gene product, GFG, in human and rat cell lysates. A polyclonal antiserum against the carboxyl terminal amino acids of rat GFG detected a single 35 kDa immunoreactive band in rat Nb2 cells (lane 6). A major 35 kDa band was also detected in the following human cell lines: lane 1, U937; lane 2, HMC-1; lane 3, CCRF-CEM; lane 4, Jurkat; lane 5, K562. Smaller bands of 17 and 28 kDa detected in the human cell lysates are also indicated.

cell lysates also contained two minor bands of 28 and 17 kDa. The 35 kDa human GFG is predicted from the open reading frame in the FGF-AS 1B sequence. The 17 kDa band may result from translation initiation at an in-frame methionine residue in exon 3 of the FGF-AS 1A mRNA. The 28 kDA species may be a proteolytic fragment of the full-length protein or it may result from translation initiation at an in-frame CUG codon in exon 2 of the FGF-AS mRNA. Immunohistochemistry was used to evaluate expression and cellular localization of FGF-2 and GFG protein in an immunoblastic lymphoma involving a lymph node (Fig. 3, panels a and c, respectively) in comparison to lymphoc-

We next examined the regulation of FGF-2 and FGF-AS mRNA in K562 cells in response to growth arrest and serum or cytokine stimulation. As shown in Fig. 4A, although FGFAS 1A transcripts were present, FGF-2 and FGF-AS 1B mRNAs were at the limit of detection following growth arrest in 0.5% serum for 24 h. Re-addition of 10% fetal bovine serum to the medium resulted in a rapid but transient increase in the FGF-AS 1B mRNA splice form, which was maximal around 15–30 min, and declined over the next several hours to basal levels. This was accompanied by a slower but sustained induction of FGF-2 mRNA expression beginning at 2.5 h and lasting for at least 12 h after serum addition before returning to basal levels at 24 h. There was little or no effect of serum treatment on the FGF-AS A mRNA splice form. Interleukin 2 (IL-2) stimulation of serum-restricted K562 cells also elicited a rapid, but transient increase in FGFAS mRNA, whereas stimulation of FGF-2 mRNA accumulation was more sustained (Fig. 4B). A similar pattern of FGF-2 and FGF-AS expression was observed in the rat Nb2 T-lymphoma cell line in response to growth arrest and stimulation with prolactin, an obligate cytokine for these cells. As shown in Fig. 4C and D, FGF and FGF-AS mRNA levels were both low in growth-arrested Nb2 cells. Similar to the IL2 response in K562 cells, the response of FGF-AS mRNA to PRL was transient, being maximally induced within 30 min following PRL stimulation, and returning to control levels within 10 h. In contrast, FGF-2 mRNA levels increased more slowly, but were sustained for up to 10 h following addition of PRL. 3.3. Expression and nuclear trafficking of FGF-2 and GFG proteins in response to IL-2 and prolactin In order to determine if the changes in sense and antisense FGF mRNAs were reflected at the protein level, we examined the effect of cytokine treatment on FGF-2 and GFG by western blot and by immunofluorescent confocal microscopy. Western blot analysis showed that K562 cells express three isoforms of FGF-2 with molecular weights of 18, 28 and 34 kDa (Fig. 5A). The FGF-2 immunoreactive bands were detected with increasing intensity in the cytoplasm of K562 cells treated with IL-2 from 6 to 24 h and this was accompanied by the appearance of FGF-2 in the cell nucleus. Nuclear FGF-2 consisted predominantly of the

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Fig. 3. Immunohistochemical staining for FGF-2 and GFG protein in an immunoblastic lymphoma involving a lymph node and in a histologically normal lymph node. Panel a: FGF-2 immunoreactivity was seen only in the cytoplasm of immunoblastic lymphoma cells (brown staining, solid arrow). Panel b: no FGF-2 immunoreactivity was seen in lymphocytes in a histologically normal lymph node. Panel c: GFG immunoreactivity was both cytoplasmic (solid arrow) and cell nuclear in immunoblastic lymphoma cells (open arrow). Panel d: cell nuclear GFG immunoreactivity (open arrows) alone was seen in half of all normal lymphocytes in a lymph node (magnification ×800).

34 and 28 kDa isoforms but also contained the 18 kDa isoform which was detected with a larger amount of cell extract (not shown). In order to confirm that the increase in nuclear FGF-2 was a result of IL-2 treatment and not simply an accumulation with time in culture, K562 cells were cultured for 24 h with or without the addition of IL-2. The addition of IL-2 markedy increased the level of both high molecular weight isoforms of FGF-2 in the nuclei of K562 cells (Fig. 5B). Rat Nb2 lymphoma cells also contained multiple FGF-2 isoforms of 18, 22 and 34 kDa in size (Fig. 5C and D). As with the human cells, FGF-2 was found primarily in the cytoplasm of quiescent Nb2 cells (Fig. 5C). Treatment with IL-2 (Fig. 5C and D) or PRL (not shown) markedly increased the abundance of the 18 kDa isoform in the cell cytoplasm, whereas the 22 and 34 kDa isoforms became detectable in nucleus at 6 and 24 h following cytokine stimulation. The increase in FGF-2 levels following cytokine stimulation was accompanied by an increase in the level of immunoreactive GFG at 3 and 6 h following IL-2 treatment (Fig. 5E). Immunofluorescent microscopy confirmed the primarily cytoplasmic localization of FGF-2 in control K562 cells cultured in the absence of IL-2 (Fig. 6A, 0 h). IL-2 treatment of these cells increased total FGF-2 immunofluorescence and induced a pronounced subcellular redistribution from cytoplasm to nucleus within 6 h of cytokine addition (Fig. 6A). Remarkably, IL-2 also stimulated cytoplasmic to

nuclear translocation of GFG which occurred by 6 h of IL-2 treatment (Fig. 6B). FGF-2 and GFG immunofluorescence remained predominantly cytoplasmic in time matched controls cultured in the absence of IL-2 for 24 h. Similarly, in quiescent Nb2 cells, immunofluorescence of both FGF-2 and GFG was predominantly cytoplasmic (Fig. 6C and D). PRL treatment of Nb2 cells induced an increase in the intensity of both FGF-2 and GFG-associated immunofluorescence, and a time-dependent nuclear translocation of both proteins by 6 h (Fig. 6C and D).

4. Discussion The present study demonstrates for the first time the coordinate expression of the FGF-2 and FGF-AS genes in hematopoietic tissues and tumor cell lines. We also demonstrate translation of the FGF-2 and FGF-AS mRNAs with colocalization of their protein products to the nucleus following serum or cytokine stimulation. The regulated co-expression of FGF-2 and FGF-AS mRNAs was unexpected. Gagnon et al. [19] had previously reported that FGF-AS transcripts were the only FGF mRNA species expressed by human lymphoid cell lines. In contrast, Krejci et al. [8,27] reported that both lymphocytic and myeloid leukemic cells express FGF2, but did not examine FGF-AS expression. We confirm that FGF-AS mRNA is the major FGF mRNA expressed in nor-

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Fig. 4. Effect of growth arrest and mitogen stimulation on FGF sense and antisense expression in human and rat leukemic cells. Panel A: effect of serum restriction and re-addition on FGF-2 and FGF-AS expression in K562 cells. Cells were growth arrested by incubation in medium containing 0.5% serum for 24 h prior to stimulation with fresh 10% FCS. Expression of FGF-2 and FGF-AS isoforms was detected by RT-PCR. Panel B: effect of IL-2 on FGF-2 and FGF-AS mRNA expression. K562 cells were cultured in 5% HS (IL-2-free) at 1 × 106 cells ml−1 . After 24 h, the cells were resuspended in fresh medium with or without the addition of IL-2 (24 U ml−1 ) for the indicated times before extraction of RNA and RT-PCR. FGF-AS transcripts were detected using the P1/P2 primer pair, and thus represent the sum of both splice forms. The lower panel depicts the mean ± S.E.M. of four independent experiments. Solid bars, FGF-2; open bars, FGF-AS. (*) Significantly different from 0 h, p < 0.05. Panels C and D: prolactin stimulation of FGF-2 (C) and FGF-AS (D) mRNA expression in rat Nb2 lymphoma cells. Growth arrested cells were treated with PRL (10 ng ml−1 ) for the indicated times. Results depict the mean ± S.E.M. of four independent experiments. (*) Significantly different from 0 h time point, p < 0.05.

Fig. 5. Expression and nuclear localization of FGF-2 and GFG proteins. Panel A and B: effect of IL-2 on expression and subcellular distribution of FGF-2 in K562 cells. The cells were cultured in 5% HS for 24 h prior to treatment with IL-2 for 6 and 24 h. Cells were pelleted for preparation of nuclear and cytoplasmic fractions. Recombinant hFGF-2 (18 kDa S.D.) was included as a positive control. Panel C and D: effect of IL-2 on FGF-2 expression in Nb2 lymphoma cells. Cytoplasmic (C) and nuclear (D) fractions are shown. Panel E: Western blot of GFG in Nb2 whole cell lysates.

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Fig. 6. Immunofluorescent detection of FGF-2 and GFG in K562 and Nb2 cells following cytokine treatment. Cells were cultured with or without IL2 (K562 cells; panels A and B) or prolactin (Nb2 cells; panels C and D) for the indicated times prior to fixation and staining as described in Section 2. K562 cells without IL-2 for 24 h served as an additional control (bottom row in panels A and B). Green fluorescence indicates either FGF-2 (panels A and C) or GFG (panels B and D). Red indicates propidium iodide (PI) staining of cell nuclei. Merged images (green plus red) are in the right hand column of each panel.

mal spleen, thymus and peripheral blood leukocytes, and in lymphoid and myeloid cell lines. However, although FGF-2 mRNA is at or below the threshold of detection by RT-PCR under basal conditions, its expression is rapidly induced following stimulation of growth arrested cells with serum or cytokines. The FGF sense and antisense RNAs are fully complementary over several hundred nucleotides at their 3 ends, and have been shown to form stable double-stranded RNA duplexes in vivo [10]. It has been hypothesized that this sense–antisense interaction contributes to the posttranscriptional regulation of FGF-2, by any one of several possible mechanisms including nuclear retention of the mRNA [28,29] or rapid degradation via inosine-specific RNAse activity [30]. The stable double-stranded RNA he-

lix may also interfere with processes such as pre-mRNA splicing, polyadenylation, RNA transport or translation initiation [31–35]. The inverse association of FGF-2 and FGFAS mRNA levels in avian, rat and human tissues, tumors and cell lines has tended to corroborate this hypothesis [14,17,23,36,37]. We have previously demonstrated that transfection and overexpression of FGF-AS in rat glioma cells results in post-transcriptional suppression of FGF-2, and inhibition of cell proliferation [9]. It is possible that the expression of the FGF-AS gene under basal conditions is sufficient to suppress FGF-2 gene expression in hematopoietic cells by transcriptional or post-transcriptional mechanisms. Our present findings support the notion that derangement of FGF-2 expression contributes to the survival and proliferative capacity of leukemic tumor cells. FGF-2 immunoreac-

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tivity was undetectable in normal lymph node, but markedly increased in primary lymphoma cells in situ. Furthermore, while the level of FGF-2 expression was low to undetectable in quiescent leukemic cells, stimulation with serum or cytokines increased FGF-2 mRNA and protein abundance, and elicited rapid nuclear accumulation of high molecular weight (HMW) FGF-2 isoforms. The nuclear actions of HMW FGF2 isoforms are associated with reduced serum requirements, anchorage-independent growth in soft agar [38–41] and acquisition of radiation resistance [42]. The induction of HMW FGF-2 seen here is consistent with the recent report by Krejci et al. [8] which detected HMW FGF-2 isoforms in all leukemia and lymphoma cell lines tested. We have previously demonstrated that in rat lymphoma cells FGF-2 inhibits apoptosis via a nitric oxide-bcl-2 mediated pathway [43]. Increased HMW FGF-2 expression may confer a significant survival advantage to leukemic tumor cells via an anti-apoptotic action. Our data demonstrate that FGF-2 and FGF-AS are coexpressed in a dynamic and specific pattern following release from growth-arrest by serum or cytokine addition. FGFAS mRNA expression was transiently induced prior to upregulation of FGF-2 mRNA expression. In all experiments, FGF-AS mRNA levels plateaued or began to decline before the more sustained increase in FGF-2 mRNA levels. These results raise a number of questions. Are the FGF-2 and FGF-AS promoters regulated by the same factors? Both are TATA-less, containing consensus SP1 and Ets recognition sequences which are active in the regulation of both genes [19,44]. The FGF-2 promoter also contains binding sites for AP1 and Egr1, which are not found in the FGF-AS promoter. In contrast, the FGF-AS promoter contains potential binding sites for transcription factors active in hematopoietic cells, but not found in the FGF-2 promoter. These include Lyf-1 and GATA sites, which are commonly found in the promoters of genes expressed in erythroid, megakaryocyte, mast and endothelial cells. GATA transcription factors play a critical role in proliferation, differentiation and maturation of erythroid and megakaryocytic cell, and may play an important role in the regulation of FGF-AS expression in these cells. Does FGF-AS regulate FGF-2 by classical antisense mechanisms? The temporal pattern of expression suggests that FGF-2 mRNA may be tonically suppressed by FGF-AS, either by direct antisense RNA interaction or by a process of promoter competition. However, our data clearly indicate that FGF sense and antisense mRNAs can stably coexist in the cytoplasm of leukemic cells under certain conditions, and that both transcripts are available for translation. Human lymphoid and myeloid cells express two alternative FGF-AS mRNAs, which appear to be differentially regulated. Whereas FGF-AS 1A predominated in normal hematopoietic tissues and in unstimulated K562 cells, FGFAS 1B was selectively induced in response to serum or cytokine stimulation. Alternative splicing of FGF-AS mRNAs has been reported in avian [14], rodent [9] and human [19]

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tissues, but the functional significance of these multiple antisense RNAs is not known. In the case of the rat, three FGF-AS RNA variants of 1060, 850 and 800 nucleotides in length are expressed on a tissue-specific basis [9]. The longest RNA is identical to the full length FGF antisense cDNA originally isolated from a rat liver cDNA library [16]. The other two transcripts arise by an alternative splicing mechanism that removes exon 2 (2) or exons 2 and 3 (2/3) of the FGF antisense sequence. At least 2 human FGF-AS RNAs (A and B) of 1069 and 1172 nts are generated by use of alternative first exons 1A and 1B [19]. The splicing events are outside the region of complementarity between the FGFAS and FGF-2 mRNAs, and are not predicted to play a role in the antisense regulation of FGF-2. Indeed, we have shown that all three rat FGF-AS transcripts are effective at suppressing FGF-2 levels in rat glioma cells [9]. However, alternative splicing of FGF-AS does have significant consequences for translation of the antisense encoded protein, GFG. Expression of human GFG has not previously been reported. Although no open reading frame initiating in exon 1A has been identified, a 35 kDa protein with homology to rat GFG is predicted from an open reading frame initiating in exon 1B. This is compatible with our observation of a 35 kDa immunoreactive species in all of the hematopoietic cell lines examined (Fig. 2C). The 28 kDa GFG-immunoreactive band we observe in human cell lines may arise by proteolyic processing of the 35 kDa form, or by alternative translation initiation from an in-frame CUG codon in exon 2 of either the A or B isoform (nucleotides 413–415 of the B isoform or nucleotides 309–311 of the A isoform). The coordinate induction of FGF-AS 1B mRNA and GFG immunoreactivity observed in K562 cells following serum or cytokine stimulation suggests that the FGF-AS 1B mRNA, rather than FGF-AS 1A, is the template for GFG translation. Alternative splicing generates analagous GFG isoforms in the rat. In that species, the full length mRNA encodes the 35 kDA isoform of GFG, whereas the 0.85 kb species, lacking exon 2, encodes a 28 kDa isoform preferentially expressed in cells of CNS origin. The 0.8 kb species, lacking exons 2 and 3, contains a frame shift and an early stop codon, resulting in a truncated product lacking the conserved nudix domain [9]. The role of GFG in hematopoietic cell function is not known. GFG/NUDT6 is a highly conserved protein belonging to the MutT/NUDIX family of nucleotide phosphohydrolases [16,19,45]. The nudix box motif is a signature sequence characteristic of a family of enzymes active on nucleotide diphosphates linked to some other moiety (x) [46]. The founding member of this family, the prokaryotic MutT protein, is responsible for removing oxidatively damaged nucleotides from the nucleotide pool [47]. At least 20 distinct human nudix-motif genes are present in the human genome, eleven of which (NUDT1-NUDT11) have been characterized to date. NUDT1 encodes hMTH1, the human homolog of the E. coli MutT protein [48]. NUDT2, -3, -4, and -11

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encode enzymes active on a range of signaling dinucleotides and the structurally unrelated diphosphoinositol polyphosphates [49–52]. NUDT 5 and NUDT 9 are NDP-sugar hydrolases with a preference for ADP-ribose [53,54]. NUDT7 is the 25 kDa subunit of the pre-mRNA cleavage factor IM required for the specific cleavage and polyadenylation of premRNA in mammals [55]. We have previously demonstrated that GFG can partially complement MutT activity, significantly reducing the rate of spontaneous mutation in MutTdeficient bacteria. Deletion of the nudix domain abrogated this activity, demonstrating that GFG is enzymatically active and may have an antimutagenic role in normal cell proliferation [13]. The high degree of amino acid identity between GFG forms from Drosophila to human suggests the sequence is constrained to conserve an essential function of this protein. As with some other members of the nudix family, this may involve removal of oxidatively damaged nucleotides from the nucleotide pool. Identification of the physiological substrate preference of GFG will provide further insight regarding its function. In summary, we have demonstrated that human myeloid and lymphoid cell lines and hematopoietic tissues express both FGF-2 and FGF-AS mRNAs, and that the expression of these complementary transcripts is coordinately regulated in response to serum or cytokine stimulation. The rapid induction and nuclear translocation of FGF and GFG following mitogenic stimulation suggests a role for both proteins in the regulation of lymphoid and myeloid tumor cell survival and proliferation.

Acknowledgements We thank Cancer Care Nova Scotia and the Nova Scotia Health Research Foundation for post-doctoral fellowship support to M. Baguma-Nibasheka and two summer studentships to M.S. Osman. We also wish to thank Zuoyu Zheng, MD for expert immunohistochemical studies on human tissues. This study was funded by grants from the Canadian Institutes of Health and the Cancer Research Society Inc.

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