Plasmids, small interfering RNAs (siRNAs) and transfection

II. MATERIALS AND METHODS

7. Plasmids, small interfering RNAs (siRNAs) and transfection

Plasmid constructs encoding wild-type Bcl-xL was described previously (Jeong et al., 2004). The NFl–GRD (Nf1 GAP-related domain) was cloned from cDNA by PCR amplification using the primers 5'-ATAGATCTACCATGGATCTCCAGACAAGAGCTACA TTTATG-3' and 5'-GTAAGCTTAACCAGTGTGTATCTGCCACAGGT-3'. The PCR products were digested with Bgl II and Hind III and then were subcloned into pEYFP-C1

vectors. The target sequences for the small interfering RNAs (siRNAs) (Genolution, Korea) were as follows: 5'-CAGTGAACGTAAGGGTTCT-3' for the NF1 gene, 5'-GCAA CATGGGAATTATGAA-3' for the SP1 gene, 5'-CAGGGACAGCATATCAGAG-3' for the BCL2L1 (Bcl-xL) gene, 5'-GGATAACACTTGTCTCTTA-3' for the FAF2 (ETEA) gene, and

5'-CCTACGCCACCAATTTCGT-3' for the nonspecific negative control. Plasmid constructs and siRNA transfections were performed in Opti-MEM (Invitrogen) using Lipofectamine 2000 (Invitrogen) and Lipofectamine RNAiMax (Invitrogen), respectively, according to the manufacturer’s instructions. For plasmid transfection, 80%-confluent cells grown in 60-mm dishes were transfected with Lipofectamine 2000 (Invitrogen). In brief, 3 ug of plasmid DNA and 5 ul of Lipofectamine 2000 (Invitrogen) were diluted separately in 500 ul of OPTI-MEM, and incubated for 5 minutes at room temperature, mixed gently. Mixtures were incubated for 20 min at room temperature and added to the cells. For transfection of siRNA, 60%-confluent cells grown in 60-mm plates were transfected with 30 to 100 nM siRNA with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.

8. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) Total RNA was isolated using TRIzol reagent (Invitrogen). 1ug of total RNA was treated with DNase I (Invitrogen), then reverse transcribed, using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) and oligo-dT primers, according to the manufacturer’s instructions. time RT-PCR was was performed on the 7500 Fast Real-time PCR System of Applied Biosystems with the SYBR Premix Ex Taq (Takara, Japan).

Primers used were as follows: the p187403 primer set (Bioneer, Korea) for the EGFR,

P238284 primer set (Bioneer, Korea) for NF1, 5'-GCGATGGCTCTGGCCAATGTG-3' and 5'-GAGAGTCTGCATGGAGTCTGCCA-3 for NF1-GRD, 5'-GTCGGATCGCAGCTTGG ATGGCCAC-3' for BCL2L1 and 5'-TGTTGCCATCAATGACCCCTT-3' and 5'-CTCCAC GACGTACTCAGCG-3' for the GAPDH gene (a relative quantification standard).

9. Chromatin immunoprecipitation (ChIP) assay

Cells were cross-linked by 1% (v/v) formaldehyde-containing medium for 10 min at room temperature. Cross-linking reaction was stopped using 125 mM glycine. Cells were washed PBS, resuspend in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0), and sonicated using a Vibracell sonicator (Sonic and Materials, Danbury, CT). After centrifuge, supernatant was diluted 10-fold with ChIP dilution buffer (1.25 % Triton X-100, 1 mM EDTA, 200 mM NaCl, 12.5 mM Tris-HCl pH 8.0), and 10% of the diluted lysate was used as input. The immunoprecipitation was carried out with 2 µg of pSP1 antibody or normal rabbit IgG (negative control) for 4 h at 4°C, followed by overnight incubation with protein A beads. The beads were washed and eluted in elution buffer (1% SDS, 0.1 M NaHCO3). The eluate was treated with 5 M NaCl and heated to 65°C for 4 h to reverse crosslinking. After RNase A and Proteinase K treatment, DNA was purified using a QIAquick PCR purification kit (QIAGEN, Inc., Valencia, CA) and was used as a template for EGFR promoter PCR amplification. PCRs were performed using human EGFR promoter-specific primers: A: 5'-GCACAGATTTGGCTCGACCTGGA-3' and 5'-GAGCGG GTGCCCTGAGGAGTTAATT-3'; B: 5'-TGGCCTTGGGTCCCCGCT-3' and 5'-AGGGCG GGAGGAGGAGGGAC-3'. PCR conditions were as follows: 95°C for 5 min, 95°C for 15 s,

65°C for 30 s, and 72°C for 34 s, for 40 cycles. PCR products were separated on a 1%

agarose gel containing ethidium bromide and visualized under UV light using the Gel Doc XR system (Bio-Rad, USA).

10. Immunoprecipitation and immunoblotting

Cultured cells were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5%

sodium deoxycholate, 0.1% SDS, and 50 mM Tris buffer, pH 8.0) containing 1 mM Na₃VO₄, 5 mM NaF, 1 mM PMSF, and protease inhibitor cocktail. 1 mg protein lysate were incubated with indicated antibody at 4°C overnight, followed by incubation with 25μL protein-A sepharose beads for 4 h (Invitrogen). Immunocomplexes bound to agarose A were washed three times in 500 μl RIPA buffer and were boiled in 2x SDS sample buffer. Precipitated proteins were analyzed by immunoblotting with the indicated antibodies. For Western blot analyses, proteins were heated at 100°C for 10 min and analyzed by SDS-PAGE on 8-12%

polyacrylamide gels. The proteins were electroblotted onto PVDF membrane (Millipore).

The membranes were blocked with 5% BSA or 5% non-fat skim milk in PBST (PBS with 0.1% Tween 20) and incubated with the indicated primary antibodies overnight at 4°C. After washing with PBST for 5 min three times, the membrane was incubated for 1 h with a secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Santa Cruz Biotechnology). The immunoblots were visualized using ECL Western blotting detection system (WEST-ZOL plus; Intron Biotechnology, Korea).

11. Cytoplasmic and nuclear fractionation

Cells were washed with PBS and lysed in lysis buffer A (20 mM HEPES, pH 7.4, 25 mM NaCl, 10 mM KCl, , 1 mM MgCl2, 1 mM EDTA, 20% glycerol, 0.1% Triton X100, 1 mM DTT, 1 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, protease inhibitor cocktail) by Dounce homogenization. Lysates fractionated by centrifugation at 1500 g for 5 min at 4°C and supernatants were used as the cytosolic fraction. Pellets were washed and re-suspended with lysis buffer B (20 mM HEPES, pH 7.4, 300 mM NaCl, 10 mM KCl, , 1 mM MgCl2, 1 mM EDTA, 20% glycerol, 0.1% Triton X100,1 mM DTT, 1 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, protease inhibitor cocktail) for 30 min at 4°C. Cell extracts were centrifuged at 14000xg for 10 min at 4°C .The resulting supernatants were collected and used as nuclear fraction.

12. Ras activation assay

Ras-GTP was detected by using a Ras-activation assay kit (Upstate Biotechnology).

Briefly, active Ras was precipitated by a GST fusion protein containing the Ras-binding domain of Raf (GST-Raf-RBD). Cells were lysed with lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, and 2% glycerol). 300 μg of total protein was incubated with Raf-1 RBD agarose at 4˚C for 1 h. Agarose beads were washed three times with 1 ml of ice-cold lysis buffer, boiled with a 2X Laemmli sample buffer, and separated on SDS-PAGE gels, followed by western blot analysis using an anti-Ras antibody.

13. Immunofluorescence cell staining

The cells were seeded onto 12 mm × 12 mm glass cover slips (Marienfeld, Germany).

After 24 hours, cells were fixed with 4% paraformaldehyde for 10 min. Then slides were subsequently washed three times with PBS and perrmeabilized with 0.5% Triton X‐100 in PBS for 10min. Blocking was done with PBS containing 5% bovine serum albumin for 30 min at room temperature. Cells were incubated with anti-phospho-SP1 antibody overnight at 4°C, and then were incubated with FITC-conjugated goat-anti-rabbit IgG antibody for 1 h at room temperature. After wash, the slides were mounted with mounting media containing DAPI (H-1200, Vectashield). Immunofluorescence images were captured and analyzed using LSM 710 confocal microscope (Carl Zeiss).

III. RESULTS

1. The EGFR expression level was closely related to the neurofibromin expression level in both normal and MPNST cells.

It has been reported that hyperexpression of EGFR is frequently observed in the NF1-associated MPNST tissues (DeClue et al., 2000; Carroll, 2012). In this study, I first aimed to elucidate the role of EGFR in tumor progression of NF1. I first examined whether the basal EGFR expression levels were different between SCs derived from normal tissues and MPNSTs. The EGFR expression levels were examined in the three SC lines; the normal human SC line (HSC) having both normal NF1 alleles (NF1^+/+), the sNF02.2 MPNST SC line containing one mutant NF1 allele and one normal NF1 allele (NF1^+/-), and the sNF96.2 2 MPNST SC line harboring a complete LOH at NF1 locus and no intact NF1 allele (NF1^-/-) (Arima et al., 2010; Sun et al., 2012). The reduced expression level of neurofibromin was detected in the sNF02.2 compare to the HSC, and no expression of neurofibromin was detected in the sNF96.2 (Fig. 7A).

I compared the EGFR expression levels among these cells and found that neurofibromin expression levels were significantly increased in the two MPNST SCs compared the normal SCs (Fig. 7A, B). Interestingly, I found that the expression level of EGFR protein was inversely related to the expression level of neurofibromin protein in the SCs tested. In order to confirm this finding, I carried out the same experiment in the NF1-associated primary cells, the normal phenotypic fibroblast cells (PC-N) and the malignant

MPNST fibroblast cells (PC-M) derived from a patient with NF1 (Fig. 6). The expression level of neurofibromin detected in the PC-M was significantly lower than that in the PC-N, while the expression level of EGFR in the PC-M was significantly higher than that in the PC-N (Fig. 7C, D). As observed in the SC lines, the expression level of EGFR protein was dependent on the expression level of neurofibromin protein in the primary cells. These results suggested that upregulation of EGFR in the NF1-associated MPNSTs may be caused by neurofibromin deficiency.

Fig. 7. Upregulation of basal EGFR expression in NF1-associated MPNST cells. Protein levels of the neurofibromin and EGFR in three Schwann cell lines (A, B) and two primary-cultured cells were determined by Western blotting. α-tubulin protein was used as the internal control. *P < 0.05 and **P < 0.01.

2. Manipulation of NF1 gene expression caused alterations in the transcriptional expression of the EGFR gene.

In order to determine if the expression level of EGFR is dependent on the expression level of the NF1 gene, I manipulated the NF1 gene expression in the primary cells and cell lines. Depletion of neurofibromin expression by short interfering RNA (siRNA) treatment for the NF1 gene caused an increase in the EGFR expression in the normal HSC and malignant sNF96.2 SCs, primary PC-N and PC-M fibroblast cells, and normal IMR90 fibroblast cells (Fig. 8A-E). Next, I performed an overexpression of NF1 gene in the sNF96.2 cells lacking intact NF1 alleles. Because the NF1 gene is very large, and the GAP-related domain of neurofibromin (NF1-GRD) is sufficient to restore normal growth in mouse Nf1^-/- cells (Hiatt et al., 2001), I used a GFP-fused NF1-GRD plasmid construct for the neurofibromin overexpression experiments instead of a plasmid construct containing the full-length NF1 gene. Overexpression of the NF1-GRD-GFP in sNF96.2 cells resulted in the decreased EGFR expression, as expected (Fig. 8F). Alterations in EGFR level by manipulating the NF1 gene expression had an influence on the Ras/Raf/Mek/Erk pathway.

Expression levels of EGFR were associated with protein levels of the activated forms of Ras GTPase, GTP-Ras, and/or its downstream effector Erk1/2, phosphorylated Erk1/2 (pErk1/2) (Fig. 8). Importantly, quantitative real-time reverse-transcribed-PCR (RT-PCR) demonstrated that both the significantly increased EGFR expression level by the knockdown of NF1 gene and the significantly decreased EGFR expression level by the overexpression of NF1-GRD were responsible for the changes in transcriptional expression of the EGFR gene (Fig. 9).

Fig. 8. Correlation between the expression levels of the EGFR and neurofibromin. (A-E) The indicated cells were transfected with siRNAs for the NF1 gene (100 nM) or the nonspecific negative control (100 nM). After 72 h incubation, protein levels of the indicated proteins were determined by Western blot analysis. α-tubulin protein level was used as the internal control. (F) sNF96.2 cells were transfected with pEGFP-C1 vector or the NF1-GRD plasmid construct and then incubated for 24 h. Protein levels of the indicated proteins were determined by Western blot analysis. α-tubulin protein level was used as the internal control.

Fig. 9. Increased transcriptional expression of the EGFR by the downregulation of NF1 expression. (A-C) The indicated cells were transfected with siRNAs for the NF1 gene (100 nM) or the nonspecific negative control (100 nM) and incubated for 72 h. (D) sNF96.2 cells were transfected with pEGFP-C1 vector or the NF1-GRD plasmid construct and then incubated for 24 h. Relative NF1 and EGFR mRNA levels in cells transfected with the control or NF1 siRNAs were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). *P < 0.05 and **P < 0.01.

3. Neurofibromin-regulated EGFR expression occurred by modulating the binding of the Sp1 transcription factor to the EGFR gene promoter

Because specificity protein 1 (Sp1) is known to be a major transcription factor of the EGFR gene (Kageyama et al., 1988), I aimed to examine if the neurofibromin-mediated regulation of transcriptional expression of the EGFR gene was influenced by Sp1. At first, I tested whether basal transcriptional expression of the EGFR gene is regulated by Sp1 in SCs.

The HSC and sNF96.2 SCs knocked down of SP1 by siRNAs treatment showed decreased expression of EGFR (Fig. 10). Next, I examined whether alterations in the NF1 expression level affect the Sp1 expression and/or activation. Knockdown of NF1 by siRNAs revealed increased Sp1 expression level and increased protein level of the activated form of Sp1, phosphorylated Sp1 at Threonine residue 453 (pSp1) (Milanini-Mongiat et al., 2002; Chu and Ferro, 2005), in the all tested cells, including HSC and sN02.2 SCs, primary PC-N and PC-M cells, and normal fibroblast IMR90 cells (Fig. 11A-E), while overexpression of NF1-GRD in sN96.2 SCs resulted in decreased Sp1 and pSp1 (Fig. 11F).

Fig. 10. The Sp1-mediated expression regulation of the EGFR gene. HSC (A) and sNF96.2 (B) cells were transfected with siRNAs for the SP1 gene (30 nM) or the nonspecific negative control (30 nM). After 72 h incubation, protein levels of the Sp1 and EGFR proteins were determined by Western blot analysis. α-tubulin protein level was used as the internal control.

Fig. 11. Alterations in the total Sp1 and phosphorylated Sp1 levels in NF1-manipulated cells. (A-E) The indicated cells were transfected with siRNAs for the NF1 gene (100 nM) or the nonspecific negative control (100 nM). After 72 h incubation, protein levels of the Sp1 and phosphorylated Sp1 (pSp1) were determined by Western blot analysis. α-tubulin protein level was used as the internal control. (F) sNF96.2 cells were transfected with pEGFP-C1 vector or the NF1-GRD plasmid construct and then incubated for 24 h. Protein levels of the Sp1 and pSp1 were determined by Western blot analysis. α-tubulin protein level was used as the internal control.

Because nuclear localization of the activated pSp1 (Davie et al., 2008; Ito et al., 2009) is known, we investigated the nuclear-localizing pSP1 protein levels by manipulating the NF1 gene expression. Knockdown of NF1 by RNAi in HSC and IMR90 cells showed the

increased pSp1 in the nucleus, while overexpression of NF1-GRD in sN96.2 cells showed the reduced pSp1 in the nucleus (Fig. 12). Next, in order to prove the hypothesis that neurofibromin regulates EGFR expression at the mRNA level via modulating transcriptional activity of Sp1 which is influenced by phosphorylation at Threonine residue 453, I tested whether there were alterations in pSp1 binding to EGFR gene in response to neurofibromin expression level. Downregulation of NF1 in HSC and IMR90 cells resulted in the elevated pSp1 level in the nucleus rather than cytosol (Fig. 13 A, B).

To examine whether the increased pSp1 in the nucleus leads to the increased binding of pSp1 to EGFR promoter, I preformed the site-specific chromatin immunoprecipitation (ChIP) targeting the pSp1-binding site of EGFR gene as described the previous report (Schuch et al., 2012). After pulling-down the pSp1-assocaited chromatins using anti-pSp1 antibody in the HSC cells treated NF1 or negative control siRNAs, PCR amplification for the two pSp1 binding-regions on the EGFR promoter was performed. The results of site-specific ChIP on HSC cells revealed that binding of pSp1 to EGFR gene was stimulated by knockdown of NF1 (Fig. 13C). These results demonstrated that neurofibromin regulates EGFR expression via modulating the binding of Sp1 transcription factor to the EGFR gene promoter.

Fig. 12. Nuclear localization changes of the phosphorylated Sp1 in NF1-manipulated cells. HSC and IMR 90 cells were transfected with siRNAs for the NF1 gene (100 nM) or the nonspecific negative control (100 nM) and then incubated for 72 h. sNF96.2 cells were transfected with pEGFP-C1 vector or the NF1-GRD plasmid construct and then incubated for 24 h. Cells were further incubated with anti-phospho-SP1 (pSp1) antibody overnight at 4°C, and then were incubated with FITC-conjugated goat-anti-rabbit IgG antibody for 1 h at room temperature. After wash, the slides were mounted with mounting media containing DAPI for staining the nucleus. Immunofluorescence images were captured and analyzed using LSM 710 confocal microscope.

Fig. 13. Neurofibromin-regulated EGFR expression via modulating the binding of the Sp1 transcription factor to the EGFR gene promoter. HSC (A) and IMR90 (B) cells were transfected with siRNAs for the NF1 gene (100 nM) or the nonspecific negative control (100 nM). After 72 h incubation, the cells were harvested and homogenized. Total cell extract were divided into cytosolic and nuclear fractions. Protein levels of the phosphorylated Sp1 (pSp1), nuclear marker Lamin, and α-tubulin proteins were assessed by Western blot analysis.

Lamin and α-tubulin protein level was used as each fraction marker. (C) The site-specific chromatin immunoprecipitation targeting the pSp1-binding site of EGFR gene. HSC cells were transfected with siRNAs for the NF1 gene (100 nM) or the nonspecific negative control (100 nM). After 72 h incubation, nuclei were lysed, and chromatin was fragmented by enzyme digestion. Chromatin was immunoprecipitated either by rabbit IgG antibody as a negative control or anti-pSp1 antibody. Non-immunoprecipitated chromatin was used as total input. DNAs form Input and IgG and SP1-immunoprecipitated chromatins were subjected to PCR analysis. PCR was performed using the primers corresponding to each region (regions 1 and 2) of the promoter region of EGFR gene. Structure of the 5′-region of the EGFR gene is shown in the bottom.

4. Neurofibromin-regulated EGFR expression occurred by modulating the Erk/Sp1-signaling pathway

Next, I aimed to clarify the molecular mechanisms of how alterations in neurofibromin expression in SCs modulate EGFR expression level. Extracellular regulated kinase (Erk)1/2 is reported to be one of the key protein kinases that are responsible for Sp1 phosphorylation (Merchant et al., 1999; Chupreta et al., 2000). I first tested whether inhibiting Erk1/2 could influence Bcl-xL expression level in HSC cells. When HSC cells were treated with the Erk1/2 inhibitor PD98059 for 24 h, EGFR and Sp1 protein levels decreased in a dose-dependent manner (Fig. 14A). Next, neurofibromin-depleted HSC cells following transfection with NF1 siRNAs exhibited a increase in pErk1/2, Sp1, and EGF levels in the absence of PD98059, but neurofibromin-depleted HSC cells showed a weak increase in pErk1/2 and Bcl-xL levels in the presence of PD98059 (Fig. 14B). These results suggested that the Erk1/2-mediated Sp1 level may play a crucial role in the NF1 dose-dependent EGFR expression changes.

Fig. 14. Knockdown of NF1-induced EGFR expression by activating the Erk1/2 and Sp1. (A) HSC cells were treated with DMSO (control) or the indicated concentrations of the Erk1/2 inhibitor PD98059 for 24 h. Protein levels of Erk1/2, phosphorylated Erk1/2 (pErk1/2), Sp1, EGFR, and α-tubulin were assessed by Western blotting. α-tubulin protein level was used as the internal control. (B) HSC cells were transfected with or without the NF1 siRNAs and DMSO or 70 μM PD98059, and then incubated for 24 h. Protein levels of Erk1/2, phosphorylated Erk1/2 (pErk1/2), Sp1, EGFR, and α-tubulin in the HSC cells were assessed by Western blotting. α-tubulin protein level was used as the internal control.

5. MPNST cells were more resistant than normal cells to anticancer drugs inducing apoptosis.

Understanding the mechanism of drug resistance is crucial for developing new strategies for targeted chemotherapy. To examine whether the chemosensitivity to anticancer drugs between normal and malignant Schwann cell (SC) lines was different, I investigated cytotoxic sensitivity to the representative anticancer drugs inducing apoptosis in the established cell lines, the normal human SC line (HSC), the sNF02.2 MPNST SC line, and the sNF96.2 2 MPNST SC line (Jouhilahti et al., 2011). Since Doxorubicin, Cisplatin and Etoposide have already been studied in the patients with NF1-associated MPNSTs (Kinebuchi et al., 2005; Landy et al., 2005; Kroep et al., 2011; Moretti et al., 2011), I used these three anticancer drugs in this study. Cell viability assay results showed that the sNF02.2 and sNF96.2 MPNST cells were more resistant to all three drugs than normal HSC cells (Fig. 15A-C). Next, I tried to confirm this result in the NF1-associated primary cells. As observed in the cell lines, the primary MPNST cells (PC-M) were more resistant to all three drugs than the primary normal phenotypic cells (PC-N) (Fig. 15D-F).

Fig. 15. Comparison of the chemosensitivity to anticancer drugs between normal cells and MPNST cells. (A, B, C) Established normal human Schwann cell line (HSC), NF1-associated MPNST cells (sNF02.2) and human NF1-depeted Schwann cell line (sNF96.2) and (D, E, F) primary tissue cultured normal cells (PC-N) and MPNST cells (PC-M) were treated with the indicated concentrations of Doxorubicin, Cisplatin or Etoposide. After 24 h incubation, cell viability was determined by Ez-Cytox assay.

6. The downregulation of NF1 expression reduced the apoptosis sensitivity to Doxorubicin in normal cells.

Because the NF1-assocaited MPNST cells showed reduced apoptosis sensitivity to anticancer drugs, I examined whether depletion of neurofibromin by siRNAs treatment targeting the NF1 gene had an influence on apoptotic cell death by anticancer drugs. The

문서에서 The Role of Bcl-xL and EGFR in the Malignant Progression of Neurofibromatosis type 1 (페이지 30-0)