CHD5, A Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas

Tomoyuki Fujita; Jun Igarashi; Erin R. Okawa; Takahiro Gotoh; Jayanthi Manne; Venkatadri Kolla; Jessica Kim; Huaqing Zhao; Bruce R. Pawel; Wendy B. London; John M. Maris; Peter S. White; Garrett M. Brodeur

Disclosures

J Natl Cancer Inst. 2008;100(13):940-949. 

In This Article

Subjects and Methods

The 101 samples (91 from the Children's Oncology Group [COG] and 10 from the Children's Hospital of Philadelphia [CHOP]) that were used in this study were chosen to be representative of rigorously defined clinical and biologic risk groups that were consistent with those used for COG but included additional genetic variables. The CHOP Institutional Review Board approved this study, and written informed consent was obtained before sample collection. The patient population is described in more detail elsewhere.[23] Briefly, for this study, low-risk patients were defined as infants (<1 year of age) with stage 1 or 2 disease by the International Neuroblastoma Staging System (INSS)[24] and favorable biologic features. Intermediate-risk patients were almost all patients with INSS stage 3 disease who were older than 1 year. High-risk patients were defined as those with INSS stage 3 or 4 disease (only two had stage 3 disease) who were older than 1 year. High-risk patients were divided into two subsets: those without MYCN amplification (high risk) and those with MYCN amplification (ultrahigh risk). RNA was obtained from these 101 neuroblastoma tumors and subjected to microarray expression profiling using Affymetrix (Santa Clara, CA) U95A chips, as described.[23] Twelve of the 23 genes from the SRD were present on these chips (AJAP1, NPHP4 KCNAB2, CHD5, RPL22, ICMT, ACOT7, HES2, TNFRSF25, KLHL21, THAP3, and CAMT1).

Tumor specimens from newly diagnosed patients were submitted between September 1, 1993, and May 30, 2003, and the median follow-up time of patients who did not experience an event was 3.4 years. More specifically, 41 patients were younger than 1 year and 60 were 1 year or older; 27 were stage 1 according to INSS, one patient was stage 2, 23 were stage 3, and 50 were stage 4. Of these 101 patients, 20 had MYCN amplification, 48 had favorable histology, 66 were hyperdiploid, 26 had 1p deletion, and 40 had 11q deletion. Twenty-eight were low-risk, 21 were intermediate-risk, 32 were high-risk (without MYCN amplification), and 20 were ultrahigh-risk (with MYCN amplification). Survival data were available for 99 of the 101 patients.

Neuroblastoma cell lines NLF, IMR5, SK-N-SH, SK-N-FI, CHLA-51, CHLA-79, CHLA-90, CHLA-123, CHLA-150, CHP-134, CHP-901, CHP-902R, KCN, KCNR, LA-N-5, LA-N-6, NB69, NBL-S, NGP, NMB, SK-N-AS, SK-N-BE2, SK-N-DZ, and SMS-KAN were obtained from the CHOP cell line bank and grown in vitro under standard conditions.[10,22,25] We analyzed the neuroblastoma cell lines for promoter methylation: two with 1p deletion (NLF and IMR5) and two without (SK-N-SH and SK-N-FI). These cell lines were also used to study the effect of exogenous CHD5 expression on in vitro growth, clonogenicity, and tumorigenicity. Cell growth in vitro was assessed by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A multiwell scanner was used to measure the absorbance at 570–630 nm dual wavelengths. The untreated controls were assigned a value of 100%. Apoptosis was assessed by annexin V antibody and propidium iodide staining (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer’s instructions as described previously.[26] Apoptosis was then monitored by flow cytometry analysis with a FACScan using CELLQuest software (Becton Dickinson, Mountain View, CA). For the studies of CHD5 re-expression using 5-aza-2-deoxycytidine, we plated 1–5 x 105 cells in 6-well plates, grew cells in the absence or presence of 5-aza-2-deoxycytidine (0.1–1.0 µM), and assessed cell morphology (cell size, shape, and neurite outgrowth), cell number measured by MTT assay, and CHD5 expression by real-time reverse transcription–polymerase chain reaction (RT-PCR) after 0, 1, 4, and 7 days.

Genomic DNA and total RNA were isolated from the 24 neuroblastoma cell lines above, including NLF, IMR5, SK-N-SH, and SK-N-FI, using DNeasy and RNeasy kits (Qiagen, Valencia, CA), respectively. We analyzed CHD5 expression of these 24 lines by RT-PCR using CHD5 primers provided by the manufacturer (Hs00395930_m1) and the TaqMan Applied Biosystems 7900HT real-time RT-PCR system (Applied Biosystems Inc. (ABI), Foster City, CA). Expression of the glyceraldehyde-3-phosphate dehydrogenase gene using ABI primers (Hs4333764-famMGB) was used for normalization. We isolated RNA from the same 101 primary neuroblastoma samples described above and previously.[23] We used a commercially available panel of mRNAs that were derived from 17 normal tissues from Clontech (Mountain View, CA), and fetal brain RNA (Clontech) was used as a positive control. Expression profiling was performed on the Affymetrix U95Av2 microarray,[23] and the data are freely available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). We measured differential gene expression based on 1p36 allelic status with the Patterns from Gene Expression (PaGE) algorithm.[27]

CHD5 protein expression in NLF and IMR5 parental and transfected lines was detected by immunoblotting with rabbit polyclonal anti-CHD5 antiserum (1 : 5000; Strategic Diagnostics, Inc., Newark, DE) and detected with donkey anti-rabbit polyclonal antiserum (Amersham Biosciences, Piscataway, NJ). Briefly, nuclear pellets were vortexed several times in 0.42 M NaCl buffer with all protease inhibitors present. Samples were kept on ice at all times. Clear supernatants with nuclear proteins were collected after centrifuging extracts at 20 000 x g 10 minutes. Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA). All extracts were prepared in duplicate, and at least three independent experiments were conducted. Protein complexes were detected with CHD5 antibody and had a predicted molecular weight of 250–260 kDa (based on the amino acid composition), specifically in NLF and IMR5 cells that were transfected with CHD5 in the sense orientation.

DNA was isolated from NLF, IMR5, SK-N-SH, and SK-N-FI cells as described above after growing them in standard culture conditions for 1 week. We then treated DNA with sodium bisulfite (4 M) to convert all unmethylated cytosines to uracil, and we subsequently amplified the modified DNA using RT-PCR as described previously[28] with primers specific for modified DNA that were designed to encompass from –840 to +769 from the start of CHD5 exon 1 (primer sequences are provided in Supplementary Table 1 , available online). The amplified DNA was cloned using the TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced using automated sequencing (ABI). We made six sets of primers in the CHD5 promoter region (–840 to +769; CHD5 GenBank accession number AL031847[GenBank]) to survey for methylation status. PCR products were purified using agarose gel electrophoresis and the Qiagen purification kit (Qiagen, Valencia, CA) and were cloned using the pGEM-T Easy Vector System (Promega, Madison, WI). At least 10 colonies were selected and sequenced to determine the percent methylation for each CpG. The percent methylation was determined by the number of clones with methylation of the given region (as determined by methylation-specific sequencing) divided by 10 and expressed as a percentage. All experiments were performed in duplicate and repeated twice.

We next performed clonogenicity assays by measuring colony formation in soft agar. Neuroblastoma cell lines NLF, IMR5, SK-N-SH, and SK-N-FI were stably transfected with a CHD5 sense or antisense (AS) DNA (GenBank accession number AF425231[GenBank] ) that had been cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). Expression of CHD5 was confirmed by RT-PCR analysis. Stable clones expressing CHD5 were selected and expanded and then seeded into T75 culture flasks and grown to 60%–70% confluency. Agar (0.35%) medium was prepared by mixing regular DNA-grade agar and RPMI 1640 with 10% fetal bovine serum (FBS); 1.5 mL was added to 60 mm wells. Cells were detached by incubation in trypsin-EDTA, centrifuged at 350xg, and suspended (5 x 103 cells) in 0.25% low melting point agarose (Cambrex, Rockland, ME) that was dissolved in RPMI 1640 containing 10% FBS per well. Cells were then plated on the underlayer of 0.35% agar. Agar plates were incubated at 37°C for 3 weeks, at which time cell colonies were stained with 0.005% crystal violet (Sigma, St. Louis, MO) and scored using an inverted microscope. By convention, cell clusters measuring more than 500 µM in greatest diameter were considered a colony. Each soft agar assay was performed in triplicate and repeated at least twice.

Stable neuroblastoma clones of NLF, IMR5, SK-N-SH, and SK-N-FI cells with either sense (-CHD5) or antisense (-CHD5-AS) were suspended in Matrigel (BD Biosciences, San Jose, CA). Six-week-old female nu/nu mice (Charles River Laboratory, Wilmington, MA; n = 160 total; n = 10 in each group, two independent experiments) were given subcutaneous injections of 1 x 107 cells in the right flank. Tumor size (L x [W]2 x 0.523/1000 = x cm3) and body weight were measured once weekly. Mice were killed 5–6 weeks after injection, when the majority of mice that had been injected with tumors derived from cells transfected with CHD5-AS developed tumors that exceeded 3 cm3. Tumors were removed surgically, fixed in 10% formalin, and stained with hematoxylin and eosin for histological examination. CHD5 expression was confirmed in the neuroblastoma xenografts by RT-PCR. This project was reviewed and approved by the CHOP Institutional Animal Care and Use Committee, and mice were housed and protocols were performed in accordance with institutional guidelines. Euthanasia was performed by the administration of ketamine (150 mg/kg) and xylazine (8 mg/kg) intraperitoneally followed by cervical dislocation after deep anesthesia had been obtained.

We analyzed associations between CHD5 mRNA expression and clinical factors by application of the two-sample t test or analysis of variance. Mean values for CHD5 expression are presented with 95% confidence intervals. The distinction between high and low gene expression was based on the median value. We estimated event-free and overall survival probabilities by Kaplan–Meier analysis and compared survival distributions using a two-sided log-rank test. Time to event for event-free survival was defined as the time from diagnosis until the time of first occurrence of relapse, progression, or death, or until last contact if no event occurred. Time to event for overall survival was defined as the time from diagnosis until the time of death, or until last contact if the patient was still alive. We used Cox regression models to examine the prognostic significance of CHD5 expression, 1p deletion, and MYCN amplification. The assumption of proportional hazards was checked using visual inspection of plots of the log-cumulative hazard vs log of survival time. All statistical tests were two-sided, and P values less than .05 were considered statistically significant unless otherwise indicated.

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