Background
The type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase is a homodimeric protein that is composed of 2 extracellular α and 2 transmembranous β subunits connected by disulfide bonds. IGF-IR belongs to the insulin receptor family whose members exhibit a common structural characteristic in the form of an amino acid motif (Y
XXXYY) within the activation loop of their respective kinase domains [
1,
2]. Ligand stimulation of IGF-IR causes its dimerization and phosphorylation, and subsequent activation of downstream signaling systems. Animal models have demonstrated the physiological contributions of IGF-IR to prenatal and postnatal normal cellular homeostasis through interactions with the growth hormone [
3,
4]. Thus, basal levels of activation of IGF-IR are required for the proliferation and growth of various types of cells, tissues, and organs [
4-
7]. The critical roles of IGF-IR in early development are illustrated by the
Igf1r-null mice, which develop generalized organ hypoplasia, delayed bone ossification, and abnormalities in the central nervous system. The
Igf1r-null mice die prematurely due to lung underdevelopment accompanied by respiratory failure [
8].
In addition to its physiological roles, pathological activation of IGF-IR induces cellular transformation and protection from apoptosis-- prerequisites for the establishment and growth of malignant tumors [
9-
15]. Indeed, IGF-IR is aberrantly overexpressed in and contributes to the survival of a variety of aggressive solid tumors as well as different types of myeloid, lymphoid, and plasma cell neoplasms and, therefore, it may represent an important therapeutic target [
16-
21]. IGF-IR induces its oncogenic effects through interactions with the downstream survival effectors IRS-1/PI3K/AKT, Grb/Ras/MAPK, and JAK/STAT [
22-
25].
The mechanisms underlying increased expression of IGF-IR in cancer cells are not completely understood. For instance, only a few transcription factors have been shown to bind the
IGF-IR gene promoter (15q26.3) and modulate its activity through stimulation or inhibition. These transcription factors include Sp1, WT1, E2F1, STAT1, and EGR-1 [
26-
34].
Recently, we identified IGF-IR as a major survival molecule that interacts reciprocally with nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) in NPM-ALK-expressing (NPM-ALK
+) T-cell lymphoma, an aggressive type of cancer that frequently occurs in children and adolescents [
35-
37]. Compared with its expression in normal human T lymphocytes and reactive lymphoid tissues, the expression of IGF-IR mRNA and protein is remarkably upregulated in NPM-ALK
+ T-cell lymphoma cell lines and human tumors [
36]. Nonetheless, the mechanisms leading to IGF-IR upregulation in this lymphoma remain to be elucidated. We hypothesized that increased IGF-IR expression may be explained by transcriptional aberrancies that exist inherently in this lymphoma. Our data show that the transcription factors Ikaros isoform 1 (Ik-1) and myeloid zinc finger 1 (MZF1) have lower expressions in NPM-ALK
+ T-cell lymphoma cell lines and human tumors relative to T lymphocytes. We were able to identify sites located within the
IGF-IR gene promoter that bind Ik-1 and MZF1. Forced expression of Ik-1 and MZF1 significanty decreased the activity of the
IGF-IR gene promoter and downregulated IGF-IR mRNA and protein levels in these lymphoma cells. In addition, Ik-1- and MZF1-induced downregulation of IGF-IR was assoicated with decreased NPM-ALK
+ T-cell lymphoma viability, proliferation, migration, and anchorage-independent colony formation.
Discussion
In this paper we show that previously unidentified defects in transcriptional machinery contribute to the pathogenesis of NPM-ALK
+ T-cell lymphoma. Compared with their expression in normal human T lymphocytes, the transcription factors Ik-1 and MZF1 are markedly decreased in NPM-ALK
+ T-cell lymphoma cell lines and lymphoma tumors from patients. Substantial evidence is provided to support that Ik-1 and MZF1 possess ability to bind specific sites residing within the
IGF-IR gene promoter and inhibit its activity. In agreement with these observations, ectopic expression of Ik-1 and MZF1 in NPM-ALK
+ T-cell lymphoma cells causes remarkable downregulation of the expression of IGF-IR mRNA and protein. Also, transfection of Ik-1 and MZF1 was associated with decreased cell viability, proliferation, migration, and anchorage-independent colony formation of NPM-ALK
+ T-cell lymphoma cells, asserting the tumor-suppressing impact of Ik-1 and MZF1 in this lymphoma. It has been previously shown that Ik-1 induces tumor suppressor effects primarily in hematopoietic cellular elements [
44-
46]; nonetheless, the contribution of MZF1 to tumorigenesis is more diverse as it may induce oncogenic or tumor suppressor effects in hematopoietic and non-hematopoietic cells [
47-
52].
The
IGF-IR gene promoter is tightly regulated during mammalian development, and during the embryonic and early postnatal stages it induces the transcription of high levels of
IGF-IR mRNA, which decrease to much lower levels during growth [
53]. The
IGF-IR gene promoter is a TATA-less and CAAT-less promoter, and like other structurally similar promoters, the
IGF-IR promoter is GC-rich [
26,
27,
54]. The transcription of the
IGF-IR gene is therefore initiated from a unique site contained within an “initiator” motif similar to the ones present in the terminal deoxynucleotidyl transferase and adenovirus middle late gene promoters [
27,
28,
55].
Although levels of expression of IGF-IR during physiological and pathological conditions can be rigorously determined at the transcriptional level [
56], thus far relatively few transcription factors have been shown to be capable of binding with and regulating the expression of the
IGF-IR gene promoter. The multiple GC boxes present in this promoter form potential binding sites for members of the zinc-finger transcription factor family. In line with this notion, earlier studies using rat
IGF-IR gene promoter showed that Sp1, a zinc-finger transcription factor, binds with GC boxes located within the 5′-flanking region and one homopurine/homopyrimidine motif (CT element) in the 5′-untranslated region of
IGF-IR gene promoter to enhance its activity [
28,
29]. The
IGF-IR gene promoter also includes
cis-elements for members of the early growth response family of zinc-finger proteins including the WT1 Wilms’ tumor suppressor, which, in contrast with Sp1, downregulates the expression of
IGF-IR [
30,
31]. Indeed, increased expression of WT1 protein was associated with a reciprocal decrease in the expression of IGF-IR protein and receptor number in prostate cancer cells, and downregulation of WT1 increased IGF-IR expression in glioblastoma [
57,
58]. Albeit less extensively studied, E2F1 and EGR-1 are also implicated in the positive regulation, and STAT1 in the negative regulation of
IGF-IR gene expression [
32-
34].
In addition to the direct regulatory effects of Sp1 and WT1, several studies have elucidated indirect contributions of oncogenic and tumor suppressor proteins to the regulation of the
IGF-IR gene expression through interactions with these 2 transcription factors. In breast cancer, BRCA1 appears to suppress the
IGF-IR promoter activity, but there is no evidence to support BRCA1’s binding and direct interactions with the
IGF-IR promoter. Instead, BRCA1 most likely suppresses the activity of the
IGF-IR promoter through the sequestration of Sp1 [
59,
60]. Similarly, in breast cancer cells, the estrogen receptor enhances
IGF-IR gene promoter activity via interactions with Sp1 and WT1 [
61,
62]. Also, MCF7 breast cancer cells that express caveolin-1 demonstrate much higher levels of
IGF-IR gene promoter activity, and the effects of caveolin-1 on the
IGF-IR gene promoter were mediated through Sp1 [
63]. Furthermore, the tumor suppressor transcription factor Kruppel-like factor 6 (KLF6) activates
IGF-IR gene transcription through synergy with Sp1 [
64]. Moreover, it was found that wild-type p53 downregulates
IGF-IR gene expression and mutated p53 enhances this expression [
65-
67]. The regulatory mechanisms conferred by p53 also do not involve specific binding with the
IGF-IR gene promoter but seem to be mediated, at least partially, by protein-protein interactions between p53 and Sp1.
The
NPM-ALK chimeric oncogene plays a central role in the survival of NPM-ALK
+ T-cell lymphoma [
35]. We have previously demonstrated that NPM-ALK and IGF-IR reciprocally collaborate to sustain their high phosphorylation levels in this lymphoma [
36,
37]. Here we questioned whether NPM-ALK, similar to oncogenic proteins described above, possesses regulatory capacity pertinent to IGF-IR expression. Our results show that specific abrogation of NPM-ALK by siRNA failed to reduce IGF-IR protein or mRNA levels. Furthermore, it was previously demonstrated that chimeric oncogenes such as the Ewing sarcoma fusion proteins induce the expression of IGF-I, the primary ligand of IGF-IR [
68]. Our data indicate that endogenous and secreted IGF-I levels are most likely regulated independently from NPM-ALK. Collectively, these results suggest that the effects of NPM-ALK on IGF-I/IGF-IR signaling are mediated post-translationally through phosphorylation of IGF-IR protein [
36,
37].
We have also previously demonstrated that NPM-ALK and IGF-IR are physically associated, and it appears that this physical association, through interactions with Hsp90, enhances the stability of NPM-ALK protein [
37]. In the current study, the decrease in IGF-IR expression after Ik-1 and MZF1 transfection was also associated with pronounced decrease in NPM-ALK basal protein levels. Although these results agree with our previous observations, we sought to investigate whether Ik-1 or MZF1 is capable of regulating the expression of NPM-ALK directly at the transcriptional level. The web-based transcription factor search algorithms failed to predict potential binding sites between the
NPM gene promoter, where the transcription of the
NPM-ALK chimeric oncogene is driven [
43], and either Ik-1 or MZF1. Furthermore, a luciferase assay using an
NPM reporter construct showed that transfection of Ik-1 and MZF1 does not affect
NPM promoter activity or protein levels. Therefore, our current results indicate that the decrease in NPM-ALK protein levels occurs secondarily to Ik-1- and MZF-1-induced downregulation of IGF-IR protein expression.
The Ik-1 and MZF1 transcription factors play physiological roles in the development of normal hematopoiesis [
39-
42]. In the present paper we describe for the first time in any type of cancer cells the negative regulation of
IGF-IR gene expression by Ik-1 and MZF1 transcription factors. Ik-1 regulates transcription by binding to specific consensus binding sites (C/TGGGAA/T) within target promoters [
69]. Similarly, MZF1’s 13 zinc fingers are separated into 2 arms, and each arm has the ability to independently bind to specific binding sites within the promoters of target genes: the first domain of fingers 1–4 (ZN 1–4) binds to the sequence 5′-AGTGGGGA-3′, and the second domain of fingers 5–13 (ZN 5–13) binds the core sequence 5′-CGGGnGAGGGGGAA-3′ [
41]. Similar to other transcription factors that bind with
IGF-IR gene promoter, we found that Ik-1 and MZF1 possess the potential to bind with sequences located both upstream and downstream of the transcription start site within the 5′-flanking region as well as within the 5′-untranslated region. Specifically, potential binding sites for Ik-1 are located at nucleotides −504/-491, −
138/-125, +77/+90,
+427/+440, and +1011/+1024, and potential binding sites for MZF1 are located at nucleotides −504/-496,
−299/-291,
−138/-130,
+501/+514, +919/+928, and +1011/+1019 (binding sites confirmed by ChIP are in italics). To our knowledge, these binding sites have not been previously described to bind with any of the transcription factors that are known to regulate
IGF-IR gene. Among the previously described transcription factors, Sp1, E2F1, and EGR-1 showed a greater net change in promoter activity at binding sites located downstream of the transcription start site [
29,
33,
34]. While this pattern was similar to Ik-1, MZF1 demonstrated a greater net change in
IGF-IR promoter activity at binding sites located upstream of the transcription start site, which resembles the inhibitory effects induced by WT1 [
30].
Ik-1- and MZF1-induced downregulation of IGF-IR was associated with decreased levels of its activated/phosphorylated form, pIGF-IR. These effects induced downregulation of the phosphorylation levels of the molecular targets of IGF-IR including IRS-1, AKT, and NPM-ALK. Whereas basal levels of AKT remained unchanged, the basal levels of IRS-1 decreased after transfection of Ik-1 and MZF1. To further analyze this unexpected finding, we searched the web-based transcription factor algorithms and found that the
IRS-1 gene promoter contains sites that could potentially function as targets for Ik-1 and MZF1 transcriptional activity. It is important to mention that IRS-1 is also a downstream target of NPM-ALK phosphorylation activity [
70]. Although further analysis is required to support this idea, we cannot completely rule out that Ik-1 and MZF1 act as tumor suppressors in this lymphoma through targeting the expression of IGF-IR and IRS-1.
It is important, however, to emphasize that deregulated systems underlying the pathogenesis of NPM-ALK
+ T-cell lymphoma are complex owing to the fact that they originate from more than one defected regulatory mechanism [
35]. Although our results provide strong evidence that the aberrant decrease in the expression of Ik-1 and MZF1 transcription factors contributes to upregulation of an important oncogenic protein, i.e., IGF-IR, we elected to investigate whether other transcriptional or posttranscriptional mechanisms exist to further enhance IGF-IR expression. Our experiments failed to support the presence of
IGF-IR gene amplification, an aberrant transcriptional mechanism, in NPM-ALK
+ T-cell lymphoma.
IGF-IR gene amplification has been previously reported in small subgroups of patients with solid tumors such as lung cancer and gastrointestinal stromal tumors [
71,
72]. Nonetheless, we also found that the posttranscriptional decay of
IGF-IR mRNA in NPM-ALK
+ T-cell lymphoma occurs over a remarkably prolonged time compared with the decay of
IGF-IR mRNA that is physiologically expressed in human T lymphocytes. To this end, our data suggest a model in which upregulation of IGF-IR in NPM-ALK
+ T-cell lymphoma results from multilevel defects in transcriptional and posttranscriptional mechanisms, which reflects the complexity of survival signaling in this lymphoma.
Materials and methods
Web-based transcription factor search algorithms
To identify transcription factors that can potentially bind to the human
IGF-IR gene promoter, 3 web-based transcription factor search algorithms were used: Genomatix (
www.genomatix.de), MATCH (
www.gene-regulation.com/pub/programs.html), and TFSearch (we used this transcription factor search algorithm when we initiated the study, but we noticed that now this algorithm has been removed and the website is not available for online support. Importantly, the findings we obtained from TFSearch matched exactly the findings gathered from Genomatix and MATCH).
Cell lines
Five NPM-ALK
+ T-cell lymphoma cell lines were used in this study: Karpas 299, SUP-M2, SR-786, DEL, and SU-DHL-1 (DSMZ, Braunschweig, Germany). The R
− cell line (mouse 3 T3-like fibroblasts with targeted ablation of
Igf1r gene [
8]; gift from Dr. Renato Baserga, Thomas Jefferson University, Philadelphia, PA) was used as the host cell line for luciferase assay studies. Normal human peripheral blood CD3
+ pan-T lymphocytes were used in some experiments (catalog number: PB0091F; StemCell Technologies, Vancouver, BC, Canada). In addition, Jurkat cells (ATCC, Manassas, VA) were used as a positive control for the expression of Ik-1 and MZF1 [
38,
79]. The T lymphocytes and the Jurkat and NPM-ALK
+ T-cell lymphoma cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS (Sigma, St. Louis, MO), glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in humidified air with 5% CO
2. DMEM supplemented with 10% FBS was used to culture the R
− cells under the same conditions.
Antibodies
The following antibodies were used: pIGF-IRY1131 (3021), pALKY1604 (Y664 in NPM-ALK; 3341), pAKTS473 (4051), and Ikaros (5443) (Cell Signaling Technology, Danvers, MA); IRS-1 (ab40777), AKT (ab8805), NPM (ab52644), and MZF1 (ab64866) (Abcam, Cambridge, MA); IGF-IR (396700; Life Technologies, Grand Island, NY); pIRS-1S639 (sc-22300; Santa Cruz Biotechnology, Santa Cruz, CA); ALK (M719501-2; Dako, Carpinteria, CA); c-Myc (631206, Clontech Laboratories, Mountain View, CA); IGF-I (05–172; Millipore, Billerica, MA); and β-actin (A-5316; Sigma).
Total RNA was isolated and purified using the RNAeasy Mini Kit (Qiagen). Briefly, 1 × 106 cells were collected by centrifugation at 200 g for 5 min, washed twice in phosphate-buffered saline (PBS), and subjected to lysis and homogenization with Buffer RLT using QiaShredder spin columns (Qiagen). Homogenized cells were re-suspended in an equal volume of 70% ethanol and passed through the spin columns. Cells were then washed once using Buffer RW1 and twice using Buffer RPE (Qiagen). Total RNA was collected upon elution with RNase-free water. Optical density was detected using spectrophotometry (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA).
cDNA synthesis was performed using the Superscript III RT protocol (Invitrogen, Carlsbad, CA). Approximately 0.3 μg total RNA was used for reverse transcription. Briefly, total RNA, oligo deoxy-thymine (dT), and deoxytrinucleotide triphosphate (dNTP) were admixed, and the final volume was adjusted to 10 μL using RNase-free water. The RNA mixture and primer were denatured at 65°C for 5 min and then placed on ice. The master reaction mixture consisting of 10× cDNA synthesis buffer, 0.1 M DTT, RNaseOUT, SuperscriptIII RT, MgCl2, and RNase-free H2O was prepared on ice and vortexed gently. Then, 10 μL of the reaction mixture was pipetted into each reaction tube on ice. Samples were transferred to a thermal cycler preheated to the appropriate cDNA synthesis temperature and incubated at 50°C for 60 min and then at 85°C for 5 min. Finally, 1.0 μL RNase H was added and the samples were incubated at 37°C for 20 min to remove template RNA.
Relative qPCR was used to measure the levels of IGF-IR mRNA in NPM-ALK+ T-cell lymphoma cell lines after transfection with Ik-1 or MZF1 expression vectors (Open Biosystems, Pittsburgh, PA) using reactions containing reverse-transcribed cDNA, IGF-IR primer/probe, and Taqman Mastermix (Applied Biosystems, Grand Island, NY). 18S ribosomal RNA was used as the endogenous control.
Transfection
Cells were transfected with Ik-1 or MZF1 expression plasmids using electroporation and the Amaxa 4D Nucleofector System (solution SF, program CA-150; Lonza, Walkersville, MD) and then incubated for 48 h. For luciferase assays, R− cells were transfected using Lipofectamine 2000 reagent. Briefly, 1 × 106 R− cells were seeded in 6-well plates. The following day, plasmids were incubated in 100 μL OptiMEM media for 5 min at room temperature. Simultaneously, 7 μL lipofectamine was incubated in a separate tube. Then, the contents of the plasmid tubes were added to the lipofectamine and incubated for 20 min at room temperature. Finally, the plasmid mixtures were added to the corresponding plate wells containing the R− cells. In some experiments, scrambled or ALK siRNA (Dharmacon, Pittsburgh, PA) was transfected into NPM-ALK+ T-cell lymphoma cell lines by using the same approach.
Construction of the human IGF-IR gene promoter
Three different fragments of the human
IGF-IR gene promoter were amplified using genomic DNA (Promega, Madison, WI). Briefly, 500 ng of genomic DNA was added to HotStarTaq plus Q solution additive mixture (Qiagen) and subjected to Touchdown PCR. Primers and amplification conditions are shown in Table
1 and Table
2.
Table 1
Sequence of the primers used to construct the 3 fragments (F1, F2, and F3) of the human
IGF-IR
gene promoter
F1
(Forward)
| 5′-CTC TCC TCG AGC CAC TCT GGG C-3′ |
F1
(Reverse)
| 5′-CAA GAC GTG CGG AGC GGA GC-3′ |
F2
(Forward)
| 5′-TCC GCA CGT CTT GGG GAA CC-3′ |
F2
(Reverse)
| 5′-GCC CCG AAG TCC GGG TCA CA-3′ |
F3
(Forward)
| 5′-GAC TCC GCG TTT CTG CCC CTC-3′ |
F3
(Reverse)
| 5′-CTC CAC TCG TCG GCC AGA GC-3′ |
Table 2
The amplification conditions used in Touchdown PCR for the construction of 3 fragments of the human
IGF-IR
gene promoter (∞: hold)
1 | Denature | 95°C | 15 min |
2 | Denature | 95°C | 30 sec |
3 | Anneal | 70°C | 45 sec |
| | −1.0°C
*
| |
4 | Elongate | 72°C | 1 min |
Repeat steps 2–4 (15 times) |
Phase 2
|
Step
|
Temperature
|
Time
|
5 | Denature | 95°C | 30 sec |
6 | Anneal | 60°C | 45 sec |
7 | Elongate | 72°C | 1 min |
Repeat steps 5–7 (25 times) |
Termination
|
Step
|
Temperature
|
Time
|
8 | Elongate | 72°C | 5 min |
9 | Halt reaction | 4°C | 15 min |
10 | Hold | 4°C | ∞ |
PCR products were run on 1.5% agarose gel, excised, and purified using the Qiaquick Gel Extraction Kit (Qiagen). PCR products were subcloned at a 1:5 molar ratio into the pGEM vector using the TA cloning system (Promega). The ligated products were transformed using MaxEfficiency DH5α-competent cells (Invitrogen) overnight at 37°C, and positive clones were selected and verified by PCR and direct DNA sequencing. Clones containing the correct insert were amplified in ampicillin containing Luria-Bertani broth (Corning Costar, Corning, NY). Plasmids were isolated and purified using the Purelink Quick Plasmid Miniprep Kit (Invitrogen). To construct reporter plasmids containing the human
IGF-IR gene promoter fragments, the pGEM plasmids and the PGL4.17 luciferase vector (Promega) were subjected to restriction enzyme digestion using ZraI/SpeI (Promega) and EcoICRI/NheI (New England Biolabs, Ipswich, MA). After digestion, DNA was ligated at room temperature using T4 DNA ligase (Promega). PCR conditions are shown in Table
3. Ligated products were confirmed by agarose gel electrophoresis and transformed using DH5α-competent cells. Positive clones were selected, subjected to plasmid isolation and purification using the Miniprep Kit, and verified by PCR and direct DNA sequencing.
Table 3
PCR conditions used for DNA ligation for the construction of the human
IGF-IR
gene promoter (∞: hold)
22°C | 30 min |
20°C | 30 min |
18°C | 30 min |
16°C | 30 min |
14°C | 30 min |
12°C | 30 min |
10°C | 30 min |
8°C | 30 min |
6°C | 30 min |
4°C | 30 min |
4°C | ∞ |
Site-directed mutagenesis and luciferase assay
Mutated human
IGF-IR luciferase reporter constructs were generated using the QuickChange II XL Site Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) and a set of primers depicted in Table
4.
Table 4
Sequences of the primers used to construct the 3 mutated fragments of human
IGF-IR
luciferase reporter (F1, F2, and F3)
F1
(Forward)
| 5′-CAA GAG CCC CAG CCG GGA GAA AGG GGA C-3′ |
F1
(Reverse)
| 5′-GTC CCC TTT CTC CCG GCT GGG GCT CTT G-3′ |
F2
(Forward)
| 5′-CAG AAA CGC GGA GCG CCG GCC ACC-3′ |
F2
(Reverse)
| 5′-GGT GGC CGG CGC TCC GCG TTT CTG-3′ |
F3
(Forward)
| 5′-GCC AGA GCG AGA GCG CCA AAT CCA GGA CAC-3′ |
F3
(Reverse)
| 5′-GTG TCC TGG ATT TGG CGC TCT CGC TCT GGC-3′ |
Luciferase assay was performed with the Dual Glo Luciferase Kit (Promega) after co-transfecting the R− cells with the reporter plasmids containing the wild-type or mutated IGF-IR promoter fragments or NPM promoter (kind gift from Dr. Qishen Pang, Cincinnati Children’s Hospital, Cincinnati, OH) along with Ik-1 or MZF1 expression plasmids using Lipofectamine 2000 (Invitrogen) for 48 h. Cells were trypsinized, washed twice with sterile PBS, and plated in a 96-well luminometer plate. An equal volume of Dual-Glo reagent was added and incubated for 10 min for cell lysis to occur. Firefly luminescence readings were obtained using a plate reader (PolarStar Omega, BMGLabTech, Cary, NC). Finally, the Dual-Glo Stop & Glo reagent was added and incubated for 10 min. Renilla luminescence readings were obtained using the above methods.
Chromatin immunoprecipitation (ChIP) assay
Because NPM-ALK+ T-cell lymphoma cells contain low levels of endogenous Ik-1 and MZF1, expression plasmids containing full-length Ik-1 or MZF1 were constructed by transferring the inserts into a c-Myc-tagged expression vector (pCMV-Myc-N; Clontech). Briefly, the original expression vectors containing Ik-1 or MZF1 as well as pCMV-Myc-N were subjected to digestion using the restriction enzymes NotI/SalI for Ik-1 or XhoI/ECORI for MZF1 (Promega). Digested products were verified by agarose gel electrophoresis and then excised and ligated using the HD InFusion system (Clontech). Ligated products were transformed using DH5α-competent cells, and positive clones were selected and verified by PCR.
ChIP assays were performed using the Pierce Agarose ChIP Kit (Thermo Scientific). Briefly, at 48 h post-transfection, cells were cross-linked using 1% formaldehyde, and cell pellets were lysed and re-suspended in a buffer containing 0.6 μL Micrococcal Nuclease (ChIP grade) and subjected to sonication on ice (Output 6; six 15 sec pulses, followed by 45 sec rest periods; Sonic Dismembrator, model 100; Thermo Fisher Scientific). Five microliters of digested chromatin was separated for the 10% input. The remaining sonicated samples were immunoprecipitated overnight at 4°C on a rocking platform using the c-Myc-Tag antibody and the provided plugged spin columns. Following overnight incubation, ChIP-grade Protein A/G Plus agarose beads were incubated for 2 h with the lysate at 4°C on a rocking platform. The samples were then washed and reverse cross-linked at 65°C for 40 min. The immunoprecipitated samples and input were eluted in a buffer containing 5 M NaCl and 20 mg/mL Proteinase K. Finally, chromatin DNA was recovered and purified using the DNA Clean-Up column and subjected to Touchdown PCR using HotStarTaq Master Mix and Q solution and a set of primers shown in Table
5. PCR products were run on 1.5% agarose gel.
Table 5
Sequences of the primers flanking potential binding sites (BS) of Ik-1 and MZF1 within the human
IGF-IR
promoter used in the RT-PCR reactions following the ChIP assay
Ik-1 BS2
(Forward)
| 5′-CGG GGG CAT TGT TTT TGG AG-3′ |
Ik-1 BS2
(Reverse)
| 5′-CGG GTT CCC CAA GAC GTG-3′ |
Ik-1 BS3 (
Forward)
| 5′-TCT TGT TTA CCA GC ATTA ACT CGC-3′ |
Ik-1 BS3
(Reverse)
| 5′-CCT CTC TCG AGT TCG CCT G-3′ |
Ik-1 BS4
(Forward)
| 5′-CGC CGC TTT GTG TGT GTC-3′ |
Ik-1 BS4
(Reverse)
| 5′-GCC GCC TCC TCC CTC A-3′ |
MZF1 BS2
(Forward)
| 5′-GCG GGG GCA TTG TTT TTG GA-3′ |
MZF1 BS2
(Reverse)
| 5′-CCG GGT TCC CCA AGA CGT G-3′ |
MZF1 BS3
(Forward)
| 5′-GCG CGT GTC TCT GTG TGC-3′ |
MZF1 BS3
(Reverse)
| 5′-GCG AGT TAA TGC TGG TAA ACA A-3′ |
MZF1 BS4
(Forward)
| 5′-GTG TGT GTC CTG GAT TTG GGA-3′ |
MZF1 BS4
(Reverse)
| 5′-GCA GAA ACG CGG AGT CAA AAT-3′ |
MZF1 BS5
(Forward)
| 5′-CGG CCC TTC GGA GTA TTG T-3′ |
MZF1 BS5
(Reverse)
| 5′-CAA GTC TCA AAC TCA GTC TTC G-3′ |
Western blotting
Cells were lysed using lysis buffer containing 25 mM HEPES (pH 7.7), 400 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 0.5% Triton X-100, 0.1 mM PMSF, 2 mM DTT, and phosphatase and protease inhibitor cocktails (Thermo Scientific, Rockford, IL). Protein concentrations were measured using the Bio-Rad protein assay, and optical density values were obtained using an ELISA plate reader (Bio-Tek Instruments, Winooski, VT). Proteins (50 μg) were resolved by electrophoresis on 8% SDS-PAGE and then transferred to PVDF membranes and probed with specific primary antibodies and then with appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Cardiff, UK). Proteins were detected using a chemiluminescence-based kit (Amersham Life Sciences, Arlington Heights, IL).
In addition, a commercially available kit (Qproteome Tissue Kit, Qiagen, Valencia, CA) was used to perform Western blot assay to measure Ik-1 and MZF1 protein levels in formalin-fixed and paraffin-embedded tissue sections from NPM-ALK+ T-cell lymphoma patients (experiments performed on archived human tissues were in accordance with the Helsinki Declaration of 1975, as revised in 1983, and approval of the Institutional Review Board was obtained prior to performing such experiments). Briefly, tissue sections mounted on glass slides were examined and tumor areas were identified and marked. Next, sections were deparaffinized, and tumor areas were excised from the slides and transferred into 1.5-mL collection tubes. β-Mercaptoethanol (6 μL) was admixed with the provided Extraction Buffer EXB Plus (94 μL) and then added to the excised tissues. Tissue tubes were incubated on ice for 5 min and then the contents were mixed by vortexing and incubated on a heating block at 100°C for 20 min. Using an oven with rotators, samples were incubated at 80°C for 2 h with agitation at 750 rpm. After incubation, tubes were cooled at 4°C for 1 min. The samples were centrifuged for 15 min at 14,000 g at 4°C. The supernatant containing the extracted proteins was collected. For quantification of protein yield, the Bio-Rad assay was used as described above.
MTS assay
Cell viability was evaluated using a CellTiter 96 AQueous One Solution Cell Proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (Promega). Cells were seeded in 96-well plates (1.0 × 104 cells/well) in 100 μL RPMI supplemented with 10% FBS. Twenty microliters of MTS reagent were added, and the cells were incubated at 37°C in a humidified 5% CO2 chamber for 4 h. Optical density measurements were obtained at 490 nm using an ELISA plate reader.
BrdU assay
Cell proliferation was measured using a BrdU assay kit (ExAlpha Biologicals, Shirley, MD). Briefly, 2.0 × 105 cells/mL were plated into a 96-well plate. BrdU (1:500 dilution) was added, and the plate was incubated for 24 h at 37°C. Cells were then fixed for 30 min at room temperature. After the cells were washed, anti-BrdU antibody was added for 1 h followed by peroxidase goat anti-mouse IgG conjugate (1:2000 dilution) for 30 min. Next, the 3,3′,5,5′-tetramethylbenzidine peroxidase substrate was added, followed by incubation for 30 min at room temperature in the dark. The acid Stop Solution was then added and the plate was read at 450 nm using an ELISA plate reader.
Cell migration assay
Cell migration was analyzed using 24-well Transwell plates with polycarbonate membranes (Corning Costar). Briefly, cells transfected with Ik-1 or MZF1 in serum-free culture medium were loaded into the upper compartment, and 500 ng/mL IGF-I (R&D Systems, Minneapolis, MN) in serum-free medium was loaded into the lower compartment. As controls, non-transfected cells in serum-free medium were loaded into the upper compartment with/without IGF-I loaded into the lower compartment. Plates were incubated for 4 h at 37°C, and cells migrating through the membrane into the lower chamber were counted using a particle counter and size analyzer (Beckman Coulter, Brea, CA).
Anchorage-independent colony formation assay
Methylcellulose (Methocult H4230; StemCell Technologies) (3.0 mL) was added to 15-mL tubes. Empty vector- (EV-), Ik-1- or MZF1-transfected cells were added in a 1:10 (v/v) ratio to the methylcellulose tubes and mixed well by gentle inversion. One milliliter of the mix was divided into 24-well plates in triplicate. Plates were placed in a humidified incubator at 37°C in 5% CO2 for 7 days. Then, p-iodonitrotetrazolium violet was added for 24 h for staining. Colonies were visualized using the AlphaImager system (Alpha Innotech Corporation, San Leandro, CA). In an additional experiment, SUP-M2 cells were incubated for 21 days, and the results were similar to those of the shorter incubations.
IGF-IR mRNA decay assay
Briefly, actinomycin D (Sigma) was dissolved in DMSO (final concentration: 1 mg/mL). Human T lymphocytes or NPM-ALK
+ T-cell lymphoma cell lines were treated with 1 μM actinomycin D and samples were collected at 0, 0.5, 1, 2, 4, and 8 h. Total RNA was isolated and purified, and cDNA synthesis was performed as described above. Absolute real-time qPCR was used to measure the levels of
IGF-IR mRNA in a 25-μL reaction by using 1 μL of the reverse-transcribed cDNA, 20×
IGF-IR Taqman gene expression assay primer/probe, and 2× Universal PCR Mastermix (Applied Biosystems). To create a standard curve, serial 10-fold dilutions (30, 300, 3000, 30,000, and 300,000 copies) of an
IGF-IR plasmid were used [
36].
Fluorescent in situ hybridization (FISH) assay to determine IGF-IR gene copy number
Human T lymphocytes and NPM-ALK+ T-cell lymphoma cells (10 × 104 cells) suspended in RPMI were pipetted into cytospin chambers. Cytospin slides were prepared (700 rpm at high acceleration for 5 min). The cytospin slides were fixed in ice cold 100% methanol, and stored at −20°C until FISH was performed.
We adopted a previously described approach to perform FISH assay and analysis [
72]. The SureFish probes and kit from Agilent Technologies were used. Briefly, 1.0 μL of
IGF-IR FISH probe (G100168R) and 1.0 μL of chromosome enumeration probe 15 (CEP15; G100543G), which identify centromere 15, were mixed gently in Agilent FISH hybridization buffer. Cytospin slides were prepared and placed in gradually increasing concentrations of ethanol (70%, 85%, and 100%), each for 1 min at room temperature. After allowing the slides to dry, 5.0 μL of probe/hybridization buffer mixture were added to the slides, and cover slips were applied. Hybridization was then accomplished by using the ThermoBrite system (Abbott Molecular, Abbott Park, IL). The slides were first incubated at 78°C for 5 min to denature the DNA, and then incubated at 37°C for 24 h. Thereafter, cover slips were removed and slides were placed and agitated in Wash buffer 1 (73°C) for 2 min. Subsequently, slides were transferred and agitated at room temperature for 2 min in Wash Buffer 2. The slides were air dried in the dark at room temperature, followed by pipetting DAPI (1.0 μg/mL in PBS). DAPI was then removed, and slides were mounted with ProlongGold antifade media (P36934, Invitrogen, Grand Island, NY) and viewed using the FV1000 confocal microscope (Olympus America, Center Valley, PA).
FISH scoring was performed in 55 nonoverlapping nuclei per slide. The means of the IGF1R gene and CEP15 copy numbers per cell, number of cells with two or fewer, three, and four or more copies of IGF1R and CEP15 signals, and IGF1R-to-CEP15 ratio were obtained.
Measurement of IGF-I levels secreted by NPM-ALK+ T-cell lymphoma cells
After transfection of scrambled or ALK siRNA for 48 h, cells were transferred to serum free medium for 24 h. The medium was then collected and concentrated using Amicon Ultra-15 centrifugal tubes (UFC900308; Millipore). ELISA assay was performed using the IGF-I cytokine kit (R&D Systems).
Statistical analysis
Statistical analysis was performed using the PRISM software (GraphPad, La Jolla, CA). Statistical significance was detected using one-way ANOVA and Bonferroni’s post hoc multiple comparisons test. P < 0.05 was considered statistically significant.
Authors’ contributions
DV designed experiments, performed research, analyzed data, and contributed to writing the paper; CVC, SA, Y-HS, GEG, AOK, PS provided essential experimental tools and analyzed data; HMA developed the conception of and supervised the study, provided essential experimental tools, designed experiments, analyzed data, and wrote the paper. The authors read and approved the final manuscript.