Cancer Research Meeting Calendar Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 4657, May 15, 2007. doi: 10.1158/0008-5472.CAN-06-4793
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bennett, K. L.
Right arrow Articles by Plass, C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Bennett, K. L.
Right arrow Articles by Plass, C.

Molecular Biology, Pathobiology, and Genetics

Tumor Suppressor Activity of CCAAT/Enhancer Binding Protein {alpha} Is Epigenetically Down-regulated in Head and Neck Squamous Cell Carcinoma

Kristi L. Bennett1, Björn Hackanson2,4, Laura T. Smith2, Carl D. Morrison3, James C. Lang2, David E. Schuller2, Frank Weber5, Charis Eng5 and Christoph Plass1,2

Departments of 1 Molecular Genetics, 2 Molecular Virology, Immunology, and Medical Genetics, Division of Human Cancer Genetics, and 3 Pathology and The Comprehensive Cancer Center, Ohio State University, Columbus, Ohio; 4 University of Freiburg Medical Center, Freiburg, Germany; and 5 Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation and Taussig Cancer Center, Cleveland, Ohio

Requests for reprints: Christoph Plass, Ohio State University, Division of Human Cancer Genetics, Tzagournis Medical Research Facility 464A, 420 W. 12th Avenue, Columbus, OH 43210. Phone: 614-292-6505; Fax: 614-688-4761; E-mail: Christoph.Plass{at}osumc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor suppressor CCAAT enhancer binding protein {alpha} (C/EBP{alpha}) is a transcription factor involved in cell cycle control and cellular differentiation. In a recent study, microarray expression profiling on head and neck squamous cell carcinoma (HNSCC) samples identified significant C/EBP{alpha} down-regulation, correlating with poor prognosis. However, the mechanisms of C/EBP{alpha} down-regulation remained elusive. C/EBP{alpha} has been previously found to provide an antiproliferative role in lung cancer, and our laboratory showed that its down-regulation involves epigenetic mechanisms. This prompted us to investigate the involvement of epigenetics in down-regulating C/EBP{alpha} in HNSCC. Here, we show that C/EBP{alpha} is down-regulated in HNSCC by loss of heterozygosity and DNA methylation, but not by gene mutation. We found a consistently methylated upstream regulatory region (–1,399 bp to –1,253 bp in relation to the transcription start site) in 68% of the HNSCC tumor samples, and DNA demethylation using 5-aza-2'-deoxycytidine treatment was able to significantly restore C/EBP{alpha} mRNA expression in the HNSCC cell lines we tested. In addition, C/EBP{alpha} overexpression in a HNSCC cell line (SCC22B) revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. In conclusion, we showed for the first time not only that C/EBP{alpha} has tumor suppressor activity in HNSCC, but also that it is down-regulated by DNA promoter methylation. [Cancer Res 2007;67(10):4657–64]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world (1). It is primarily caused by exposure to alcohol and tobacco products, which influence major pathways of cell proliferation (2). Surgery is done only in cases of locally advanced HNSCC, and following surgical treatment, many HNSCC patients are prone to relapse and ultimately systemic dissemination (3). Neck dissection followed by chemotherapy may allow substantial regional control, but very few patients achieve long-term remission (4). Therefore, early detection and better disease prediction via genetic and epigenetic biomarkers become crucial.

Epigenetic modifications, especially in the form of DNA methylation, have emerged as a relevant factor in the disease progression of HNSCC. Despite the fact that global DNA methylation screens showed relatively lower levels of promoter methylation in HNSCC as compared with other malignancies (5), several epigenetically silenced cancer genes have been described in HNSCC. These include secreted frizzled-related protein family genes (6), LHX6 (7), p16 (8), TCF21 (9), p53 (10), and members of the Fanconi anemia/BRCA1 pathway (11). However, none of the epigenetically regulated genes have been identified as poor prognostic markers in HNSCC (12).

In a recent study, microarray expression profiling was done on 40 HNSCC tumor samples to identify gene expression differences correlating with poor prognosis (13). CCAAT enhancer binding protein {alpha} (C/EBP{alpha}) was one of the genes analyzed, and the authors found 78% (31/40) of the HNSCC tumor samples had C/EBP{alpha} down-regulation compared with the expression in matched normals (13). Furthermore, a significant correlation was found between C/EBP{alpha} down-regulation and patients with extensive lymph node metastasis (13). Decreased C/EBP{alpha} expression could contribute to the tumor cells' characteristics of uncontrolled proliferation and loss of differentiation. This has already been shown in acute myelogenous leukemia (AML), where mutations in the coding region of C/EBP{alpha} have been found to relieve the protein of its ability to block cell cycle progression (14). In addition, C/EBP{alpha} provides an antiproliferative role in lung cancer (15), and we have recently shown that its down-regulation is conducted by epigenetic mechanisms (16). Therefore, we hypothesized that C/EBP{alpha} might play a role in tumor and metastatic suppression in HNSCC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient samples and cell lines. Frozen tumor tissues and adjacent normal tissue from HNSCC patients were attained from the Ohio State University Medical Center via the Cooperative Human Tissue Network. Surgery was done on all patients at the Ohio State University Medical Center. All sample collections were done according to the NIH guidelines and under a protocol approved by the Institutional Review Board of the Ohio State University. Control samples were collected from morphologically normal tissue located at least 3 cm from the tumor margin. Histopathologic evaluation was done on all samples for verification. The seven different established human HNSCC cell lines used in the study (SCC4, SCC5, SCC8, SCC11B, SCC17AS, SCC22B, and SCC25) were kindly provided by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI). Cell lines were maintained in DMEM with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin antibiotics.

Immunohistochemistry. Immunohistochemical staining was done as previously described (16), including three tissue microarrays containing HNSCC normal, tumor, or metastatic samples. Tissue microarrays contained triplicates of each primary tumor or metastatic tissue sample. The tumor array contained 48 patient samples, the metastatic tissue array contained 34 samples (shared in the tumor array), and the normal array contained 47 matching adjacent normal samples. Scoring was done using the following index: 0, no staining; 1, faint staining in <10% cells; 2, light staining in 10% to 50% of cells or medium staining in <10% cells; 3, light staining in all cells or dark staining in 10% cells; 4, medium staining in 10% to 50% of cells; 6, medium staining in all cells or dark staining in 10% to 50% of cells; and 9, dark staining in all cells. HNSCC tumor or metastatic tumor samples had to contain an index of at least two scores below its paired normal to be considered down-regulated. One tumor sample was without a matching normal for comparison, and 14 tumor/normal pairs lacked matching metastatic samples. The magnification used was 400x.

Migration assay. Approximately 400 µL of 10% FBS DMEM was plated in a 24-well plate. Millicell Culture Plate Inserts (Millipore) were placed into each well, and 5,000 or 20,000 cells were suspended in 300 µL DMEM without FBS. As a control, chambers with no cells were prepared similarly. After 20 h, the media was aspirated, and chambers washed with Dulbecco's phosphate-buffered saline (PBS). About 250 µL of 0.05% crystal violet 3.7% formaldehyde was placed in the upper and outer chambers. Cells were allowed to incubate for 30 min; then the crystal violet was removed, and the wells were washed with water. After allowing 15 min for drying, 80 µL of water was used to moisten a cotton tip applicator. The applicator was then used to rub the membrane underneath the chamber, broken close to the tip and placed in a reaction tube with 500 µL 100% methanol. Tubes were vortexed well and incubated for 30 min. About 100 µL of the methanol was taken into a 96-well plate for absorbance reading at 570 nm. Approximately 250 µL of 80% methanol was placed inside the chamber, incubated in a shaker for 30 min at room temperature, and 100 µL was again taken for absorbance at 570 nm. Percent migration was determined by the following calculation: 100 x [(absorbance migrated cells – absorbance background)/(absorbance migrated cells – absorbance background) + (absorbance nonmigrated cells – absorbance background)].

5-Aza-2'-deoxycytidine treatment. HNSCC cell lines were incubated for 96 h with 3 µmol/L 5-aza-2'-deoxycytidine (5-aza-dC; Sigma), with medium and drug changed daily. Treated cells were harvested for analysis after the procedure. Cells were suspended in TRIzol for RNA isolation or Laemmeli buffer [62.5 mmol/L Tris-Cl (pH, 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue, 300 mmol/L 2-mercaptoethanol] for Western analysis.

RNA isolation and cDNA synthesis. RNA was isolated according to the manufacturer's protocol. DNase treatment was done on 1 to 2 µg of RNA by adding 2 units of DNaseI (Invitrogen), 1 µL DNase buffer, and 0.4 µL RNase Out (Invitrogen) for 15 min at room temperature. About 1 µL EDTA was then added to the mix for 10 min at 65°C, followed by an incubation on ice for 5 min. cDNA synthesis was done by the following reaction: 2 µL random hexamers and 1 µL deoxynucleotide triphosphates (10 mmol/L) for 5 min at 65°C then 2 min at 4°C; 2 µL of 10x buffer, 4 µL MgCl2, 2 µL DTT, and 1 µL RNase Out was added for 2 min at 25°C; 100 units of SuperScript II (Invitrogen) for 50 min at 42°C; 15 min at 70°C, then transferred to 4°C.

Real-time PCR. Quantitative C/EBP{alpha} mRNA expression was measured using SYBR Green I (Bio-Rad) in an I-Cycler (Bio-Rad). Expression of glycosylphosphatidylinositol (GPI) was used as the internal control gene. I-Cycler conditions and reverse transcription-PCR (RT-PCR) primers are listed in Supplementary Data. We additionally did PCR on DNaseI-treated, "–" (without the reverse-transcriptase enzyme) RT samples to ensure that no DNA contamination was present in the RNA extract (given the fact that C/EBP{alpha} is an intronless gene). No amplification of PCR product was seen in these samples, indicating the absence of contaminating genomic DNA in the DNaseI-treated RNA extracts.

Sequenom methylation analysis. Quantitative DNA methylation analysis was done by Sequenom, Inc. About 1 µg of HNSCC patient or cell line DNA was bisulfite treated, in vitro transcribed, cleaved by RNase A, and subjected to matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry analysis to determine methylation patterns, as previously described (17).

Chromatin immunoprecipitation assay (ChIP). Chromatin immunoprecipitation (ChIP) was done according to the Upstate Cell Signaling Solutions Protocol. Quantitative PCR for the ChIP assay was done with 3 µL of ChIP eluate and primers surrounding the promoter binding sites. Promoter enrichment was assessed by normalization of threshold crossing of the samples compared with the threshold crossing of the negative control (no antibody).

Plasmid constructs and oligos. The pBABE-CEBP{alpha} construct was generously provided by Dr. Gokhan Hotamisligil (Harvard University, Boston, MA) and previously described (18). The pRS retroviral vector from Origene was used for stable silencing experiments. shRNA oligo sequences are listed in Supplementary Data. The sequence targeted for stable silencing was borrowed from a previous publication (19).

Transfections. Stable transfections were done as previously described (9). The target cells were collected, counted, and suspended in Laemmeli buffer for Western blot confirmation of silencing 120 h postinfection. For the transient silencing experiment, 1.6 x 105 C/EBP{alpha}-overexpressing SCC22B cells were plated per well in a six-well plate. The transfections were done in duplicates. The negative control transfection mix contained 100 µL EC-R buffer (Qiagen), 14.4 µL RNAifect (Qiagen), and DMEM + 10% FBS. The small interfering RNA (siRNA) transfection mix contained 4.8 µL 20 µmol/L siRNA stock, 95.2 µL EC-R buffer (Qiagen), 14.4 µL RNAifect (Qiagen), and DMEM + 10% FBS, for a final concentration of 30 nmol/L. The transfection media was left on the cells for 72 h. Then, the cells were trypsinized, counted, and suspended in Laemmeli buffer for Western analysis.

Colony formation assay. Colony formation assay was done as previously described (9).

Growth curve. Cells were counted using a Cell Coulter Counter. About 1.4 x 104 cells were plated in triplicates on six-well culture plates. Cells were then permitted to grow 3 days before taking counts, after which cells were washed, trypsinized, and counted every 24 h for 4 days. For each count, 500 µL of trypsin was added to each well for 3 min, after which the cells were suspended in 2 mL of PBS, and 500 µL was counted using a Coulter Counter.

Combined bisulfite restriction analysis. Genomic DNA (1 µg) in a volume of 50 µL was denatured by NaOH (final concentration, 3 mol/L) for 30 min at 37°C. The denatured DNA was then treated with 30 µL of 10 mmol/L hydroquinone and 520 µL of 3 mol/L sodium bisulfite at 50°C overnight. The primers used to amplify the bisulfite-treated DNA are as follows: BSCEBPregion1F 5'-TTTTGTTAGGTTTAAGGTTATTG-3'; BSCEBPregion1R 5'-AAACCCTAAAACCCCTTAAATA-3'. The PCR conditions were initiated with a denaturing step of 95°C for 10 min followed by 36 cycles of 96°C for 30 s, 59°C for 30 s, and 72°C for 30 s and were concluded with 72°C for 10 min. The PCR products were purified with a purification kit (Qiagen) and incubated with BstUI at 60°C for 4 h. Digested DNA was then size fractionated via PAGE to detect the methylation status.

Bisulfite sequencing. Promoter region of bisulfite-treated HNSCC cell lines and patient samples were analyzed for methylation as previously described (16).

Loss of heterozygosity analysis. A total of 122 HNSCC patient adjacent normal, stroma, and epithelial tumor samples were subjected to loss of heterozygosity (LOH) analysis using D19S433 and D19S245 markers. Sequencing of the PCR products showed whether the tumor sample had homozygosity, retention of heterozygosity, or loss of heterozygosity when compared with normal.

Northern blot analysis. The Northern blot analysis was done as previously described (16), but instead, with 8 µg of RNA. Six pairs of HNSCC normal/tumor patient samples (also included in the MassARRAY methylation analysis) were loaded onto the blot in order of increasing methylation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Image Quant was used to determine C/EBP{alpha} expression in tumor compared with its matching normal (which was set as 1).

Western blot analysis. The Western blots were done as previously described (16). After C/EBP{alpha} detection, the blot was stripped with Bio-Rad stripping buffer in 37°C for 5 min and reprobed with a 1:500 dilution of {alpha}-tubulin antibody (1:3,000 secondary mouse antibody). A 1:250 dilution was used for involucrin (Biomeda) and a 1:3,000 secondary rabbit.

Mutation analysis. The coding region of C/EBP{alpha} was amplified in 49 HNSCC tumor samples and 10 HNSCC cell lines using four sets of primers. Primer sequences and conditions were previously described (20). Mutation screening was done by comparing the resulting sequences with the known normal sequence on BLAT.

Animals and in vivo assay. Three- to four-week-old male nude mice (Taconic, NCRNU-M) were maintained in a pathogen-free environment. Injections were done as previously described (9). Tumors were palpable after 17 and 26 days from the 8.5 and 3 million cell injections, respectively. Volumes were calculated by the following formula: V = (L x W2){pi}/6.

Statistical analysis. The statistical significance of the results was calculated by unpaired Student's t test, and P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C/EBP{alpha} down-regulation in HNSCC. Based on the previous publication by Roepman et al. (13), which showed that C/EBP{alpha} is among the transcripts significantly down-regulated in poor-prognosis HNSCC patients, we first wanted to validate decreased C/EBP{alpha} expression levels in our HNSCC patient samples via immunohistochemistry analysis. Strong C/EBP{alpha} expression was detectable in epithelial cells in 39/47 (83%) of the normal tissue sections, but it is down-regulated in 33/48 (69%) of the tumor samples and 23/34 (68%) of the metastatic tumor samples (Fig. 1A and Supplementary Table S1). About 52.2% (12/23) of the paired metastatic and tumor samples shared similar levels of down-regulation, 43.5% (10/23) of the metastatic samples showed even greater down-regulation than the matched tumor samples, and 4.3% (1/23) of the tumors showed greater down-regulation than the matched metastatic samples (Supplementary Table S1).


Figure 1
View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Immunohistochemistry and DNA methylation analysis on HNSCC samples. A, tissue microarrays contained tumor (T), adjacent normal (N), or metastatic tumor (M) tissues stained with C/EBP{alpha} antibody (sc-61). Positive staining is evidenced by brown nuclear staining. Background cytoplasmic staining is negative. A representative sample is shown. Magnification, x400. B, quantitative DNA methylation analysis using MassARRAY in three amplicons spanning from –1,450 to –1,120, –580 to –297, and 164 to 379 bp relative to the transcription start site is indicated by the heat map. Arrow, transcriptional start site; box labeled C/EBP{alpha}, C/EBP{alpha} coding sequence. Slanted lines beyond the arrow, the entire exon is not shown. Each column in the heat map represents a CpG site or combination of CpG sites. Each row represents one sample. Top, five adjacent normal tissue samples (N); middle, 30 tumor samples (T); the bottom group (C) represents six HNSCC cell lines (SCC25, SCC17AS, SCC11B, SCC8, SCC5, and SCC4 in descending order). Colors representing the degrees of methylation (from 0% to 100%) are as indicated by the key. Gray squares, unanalyzable CpG dinucleotides. *, **, and *** correlate with indicated tumor samples in (C). C, bisulfite sequencing analysis for HNSCC tumors and their matching normal tissue DNAs. Arrow, transcription start site; the diagram is drawn to scale (spanning –1.5 kb upstream of the transcriptional start site). The region tested for methylation via bisulfite sequencing is indicated on this diagram (spanning from –1,423 to –1,121 bp). Bisulfite sequencing was done by comparing the sequences with 100% and 0% methylated DNA sequence (i.e., CG or TG at CpG sites, respectively). {circ}, unmethylated CpGs; bullet, methylated CpGs. Each row represents an individual clone. *, **, and *** correlate with indicated tumor samples in (B).

 
C/EBP{alpha} deletion in HNSCC. We next investigated possible ways for C/EBP{alpha} down-regulation, first evaluating the genetic mechanisms. We assessed the incidence of deletions in this genomic region in laser-captured microdissected (LCM) HNSCC samples by using markers D19S245 (200 kb downstream of C/EBP{alpha}) and D19S433 (2 Mb upstream of C/EBP{alpha}). This analysis revealed LOH in 30/49 (61%) of HNSCC patient tumor epithelium samples at D19S433, 41/60 (68%) LOH at D19S245, and 8/29 (28%) LOH at both markers. Interestingly, the highest LOH incidence was attained from D19S245, which is the marker closest to the C/EBP{alpha} locus (Supplementary Table S2).

C/EBP{alpha} mutations in HNSCC. C/EBP{alpha} mutations have been associated with increased proliferation of myeloid precursors in AML by creating a truncated isoform lacking the transactivator domain (20). Therefore, we investigated the possibility of C/EBP{alpha} mutations in HNSCC. Mutation screening was accomplished via sequencing the C/EBP{alpha} coding region from 19 HNSCC tumor samples, 10 HNSCC cell lines, and 30 LCM samples (which had also been included in the LOH analysis). Samples included in the mutation analysis were generally those that had no promoter methylation (see below) and were from five different anatomic regions encompassed by HNSCC: pharynx, larynx, oral cavity, adenoids, and tongue. Mutation analysis on these samples revealed no mutations (Supplementary Table S3).

C/EBP{alpha} methylation from –1,399 bp to –1,253 bp in HNSCC tumors and re-expression with 5-aza-dC treatment. Recently, our laboratory has shown that C/EBP{alpha} is epigenetically regulated in lung cancer (16). Therefore, we investigated epigenetic regulation of C/EBP{alpha}, a gene associated with a dense CpG island, in HNSCC. To first locate the region of methylation, we required a broad methylation analysis of the upstream and promoter-proximal regions of C/EBP{alpha}.Therefore, we did quantitative DNA methylation analysis by using the MassARRAY technique that combines in vitro transcription, RNase A cleavage, and MALDI-TOF mass spectrometry on HNSCC DNAs (17). This strategy provided information on upstream region 1 (–1,450 to –1,120 bp), promoter-proximal region 2 (–580 to –297 bp), and promoter-associated region 3 (+164 bp to +379 bp). All three of these regions are encompassed within a CpG island (i.e., the sequence contains a GC content of ≥50% over a length >200 bp). The MassARRAY analysis of region 1 showed an average of 25% methylation in the 28 HNSCC tumor samples, an average of 10% methylation in the five HNSCC adjacent normal samples, and an average of 87% methylation in the six HNSCC cell lines. Lower methylation seen in the HNSCC tumor samples compared with the HNSCC cell lines is most likely due to the potential for contaminating normal tissue in the tumor specimens. Virtually no methylation was seen in regions 2 and 3 (Fig. 1B). Interestingly, the sequence of the methylated region 1 is extremely conserved between humans, mice, rats, dogs, and opossums according to University of California–Santa Cruz Genome Browser on the March 2006 assembly. This sequence may provide promoter or enhancer activity, which is supported by previous promoter studies that have revealed increasing promoter activity by including more upstream sequence (16, 21).

Subsequent to the broad methylation analysis, we desired to further define where DNA methylation is occurring in this region. Although MassARRAY allows a large-scale methylation analysis, information on some CpG sites is lost due to the nature of the technique (i.e., cleavage products of the same size are indistinguishable from one another). In contrast, bisulfite sequencing provides methylation data on each CpG within the sequenced region. Therefore, we did bisulfite sequencing on four tumor samples (and their adjacent normals) that had shown methylation in the MassARRAY analysis. Bisulfite sequencing revealed consistent methylation in all four tumor samples in upstream region 1 not seen in their corresponding normal tissues (or "germ line DNA"; Fig. 1C). These tumor samples showed decreased C/EBP{alpha} mRNA expression (compared with adjacent normal tissue DNAs) by quantitative RT-PCR (data not shown). Furthermore, two of the four samples had been included on the tissue microarray for the immunohistochemistry analysis, which revealed significant C/EBP{alpha} down-regulation in these samples (Supplementary Table S1). This bisulfite sequencing data closely correlates with the broad methylation analysis (Fig. 1B), and it reveals that methylation exists only in upstream region 1.

To correlate the incidence of LOH and methylation, 23 samples that exhibited LOH at D19S245 (the marker closest to the C/EBP{alpha} locus) were also tested for C/EBP{alpha} promoter methylation in the upstream region 1. Combined bisulfite restriction analysis revealed methylation in 65% of these samples (15/23; data not shown). Bisulfite sequencing was done on five of these tumor samples, which showed partial methylation ranging from ~15% to 50% (Supplementary Fig. S1).

We next wanted to verify if C/EBP{alpha} promoter methylation is capable of decreasing C/EBP{alpha} expression. We did Northern analysis on six normal/tumor HNSCC patient pairs that had shown increasing methylation according to the MassARRAY analysis. Quantification of the Northern analysis revealed a strong correlation between increasing promoter methylation and decreased mRNA expression (Fig. 2A ). Quantitative RT-PCR on 18 normal/tumor HNSCC patient pairs also revealed a correlation between increasing methylation (according to the MassARRAY) and decreased C/EBP{alpha} mRNA expression (Supplementary Fig. S2). Three HNSCC cell lines that all exhibited high amounts of methylation in the upstream C/EBP{alpha} sequence (SCC11B, SCC22B, and SCC25; Fig. 1B) were exposed to 3 µmol/L of the demethylating drug 5-aza-dC for 96 h to see if demethylation allows C/EBP{alpha} re-expression. After treatment, cells were harvested and analyzed for changes in C/EBP{alpha} methylation and expression compared with the untreated cells. Bisulfite sequencing on SCC17AS cells before and after 5-aza-dC treatment revealed a decrease in upstream C/EBP{alpha} methylation (93–56%; Fig. 2B). In addition, quantitative RT-PCR and Western blot analysis revealed an increase in C/EBP{alpha} expression. SCC11B, SCC22B, and SCC25 all showed a significant increase in C/EBP{alpha} mRNA expression (P < 0.018, P < 0.008, and P < 0.001, respectively; Fig. 2C), and Western blot analysis on two HNSCC cell lines (SCC11B and SCC17AS) showed an increase in C/EBP{alpha} protein expression (Fig. 2D), which suggests epigenetic control of the gene.


Figure 2
View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. C/EBP{alpha} expression analysis for methylated HNSCC patient samples and cell lines. A, Northern analysis was done on six HNSCC normal/tumor patient pairs. Left to right, samples were loaded on the blot in order of increasing methylation (according to MassARRAY). Top, hybridization with C/EBP{alpha} probe; bottom, GAPDH. Quantification by ImageQuant of the C/EBP{alpha} mRNA expression is shown beneath each lane. Each sample was normalized to its GAPDH, and the C/EBP{alpha} expression of each tumor was normalized to its matching normal, which was set as 1. The average methylation according to MassARRAY is listed below the expression quantification. B, bisulfite sequencing analysis was done on SCC17AS cells with and without 5-aza-dC. The diagram is drawn to scale; arrow, transcription start site; the region tested for methylation via bisulfite sequencing is indicated on this diagram (spanning from –1,423 to –1,121 bp). Bisulfite sequencing was done by comparing the sequences with 100% and 0% methylated DNA sequence (i.e., CG or TG at CpG sites, respectively). {circ}, unmethylated CpGs; bullet, methylated CpGs. Each row represents an individual clone. C, RT-PCR analysis was done on RNA isolated from the treated or untreated HNSCC cells (SCC11B, SCC22B, and SCC25), subjected to DNase treatment and cDNA synthesis. Each C/EBP{alpha} expression level was normalized to its internal control (GPI expression). The SCC cell line with the lowest endogenous C/EBP{alpha} expression, SCC25, was set to 1 for comparison. *, P < 0.0177; **, P < 0.0083; ***, P < 0.0010. D, Western analysis was done on SCC11B or SCC17AS cells with and without treatment. Cells were isolated, suspended in Laemmeli buffer, and loaded on a 12% PAGE gel. Upon transfer, the blot was probed with C/EBP{alpha} (sc-61) and {alpha}-tubulin (control for loading) antibodies.

 
C/EBP{alpha} overexpression provides tumor suppressor activity in vitro and in vivo in HNSCC. To assess the potential of C/EBP{alpha} for tumor suppressor activity in HNSCC, we overexpressed C/EBP{alpha} in a HNSCC cell line (SCC22B). After protein overexpression was confirmed via Western analysis (Fig. 3A ), growth curve analysis and colony formation assays were done. In comparison to the pBABE-transfected cells and SCC22B untransfected cells, C/EBP{alpha} transfectants exhibited a significant decrease in proliferation (21 h versus 50 h average doubling time, respectively) and colony formation (Fig. 3B and C). Next, we used a migration assay to investigate the ability of C/EBP{alpha} to inhibit metastasis. The migration assay showed a significant decrease in migration of C/EBP{alpha} transfectants compared with the pBABE transfectants (Fig. 3D).


Figure 3
View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Analysis of C/EBP{alpha} tumor suppressor activity in an HNSCC cell line (SCC22B). A, the Western blot contained lysates from untransfected SCC22B cells (negative controls) and C/EBP{alpha}-transfected SCC22B cells. A total of 5 µL (50,000 cells), 15 µL (150,000 cells), and 30 µL (300,000 cells) of each lysate were loaded. Equal loading was confirmed by {alpha}-tubulin. B, 1 x 104 SCC22B untransfected, pBABE-transfected, or C/EBP{alpha}-transfected cells were plated in triplicates. Cells were permitted to grow 3 d after plating before taking the first count (day 1). Growth was assessed by counting the cells every 24 h for 4 d. ***, P < 9.39 x 10–6. C, colony formation was compared between C/EBP{alpha}-overexpressing SCC22B cells, pBABE-transfected SCC22B cells, and untransfected SCC22B cells. P < 0.002738. D, cell migration was compared between C/EBP{alpha}-overexpressing SCC22B cells and pBABE-transfected SCC22B cells. *, P < 0.018.

 
To validate the in vitro tumor suppressor activity, we did in vivo analysis using nude mice. We executed two separate in vivo experiments using 3 and 8.5 million cells for the injections. The experiment using 3 million cells yielded 6 out of 10 mice with tumors from the wild-type SCC22B cells, and one of these also had a tumor from the C/EBP{alpha}-pBABE–transfected SCC22B cells (data not shown). We did immunohistochemistry on the tumors of this mouse that formed tumors from both cell types. As expected, this analysis revealed increased C/EBP{alpha} expression in the tumor from the C/EBP{alpha}-overexpressing SCC22B cells (Fig. 4A ). The experiment using 8.5 million cells yielded 10 out of 10 mice with tumors from the pBABE-transfected SCC22B cells. However, none of these mice formed tumors from the C/EBP{alpha}-pBABE–transfected SCC22B cells (Fig. 4B and C).


Figure 4
View larger version (55K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. C/EBP{alpha} overexpression in vivo. A, the tumors analyzed were from the only mouse in a previous experiment that developed tumors from both cell types (SCC22B cells and SCC22B cells overexpressing C/EBP{alpha}). Tumors are shown to scale adjacent to their immunohistochemistry. Tumor tissues were stained with C/EBP{alpha} antibody (sc-61). Positive staining is evidenced by brown nuclear staining. Background cytoplasmic staining is negative. Magnification, x400. W, pBABE-transfected SCC22B; C SCC22B cells overexpressing C/EBP{alpha}. B, the 10 mice injected with pBABE-transfected SCC22B cells (left flank) and C/EBP{alpha}-overexpressing SCC22B cells (right flank). C, the average tumor volume of the SCC22B cells versus the average tumor volume from the SCC22B cells overexpressing C/EBP{alpha} is plotted. Volumes are plotted from 18 to 38 d postinjection. Volumes were calculated by the following formula: V = (L x W2){pi}/6 (45). *, P < 0.009385.

 
C/EBP{alpha} re-expression allows differentiation in HNSCC. One of the main functions of C/EBP{alpha} is cellular differentiation. With differentiation-inducing agents, C/EBP{alpha} expression has been shown to increase in keratinocytes (22). Keratinocytes are a major cell type of the epidermis and account for ~90% of epidermal cells, allowing them to serve as the normal cell population control for HNSCC. Increased C/EBP{alpha} expression leads to elevated involucrin expression (a squamous cell differentiation marker in keratinocytes; ref. 22). Therefore, to check whether demethylation and increased C/EBP{alpha} expression induces differentiation, we used an involucrin antibody that showed increased involucrin expression subsequent to demethylation treatment. Therefore, this analysis suggests a correlation between increased C/EBP{alpha} expression and increased involucrin expression (Fig. 5A ). Furthermore, C/EBP{alpha} binding within the involucrin promoter was previously found to be required for its transcription (23), so we investigated whether increased involucrin expression is a direct effect from C/EBP{alpha} re-expression. To do this, we did ChIP analysis on SCC22B and SCC17AS cells with and without 5-aza-dC treatment. Quantitative PCR was then done on the ChIP eluate, using primers surrounding the C/EBP{alpha} binding site in the involucrin promoter. This revealed a significantly greater amount of involucrin promoter pull-down following 5-aza-dC treatment (Fig. 5B). This suggests that C/EBP{alpha} re-expression allows C/EBP{alpha} to bind to the involucrin promoter and activate its transcription. Although we did not exclude the possibility for methylation of involucrin, it is very unlikely that it is directly controlled by methylation [since it contains very few CGs (8 in 1,500 bp upstream of the TSS) and lacks a CpG island].


Figure 5
View larger version (54K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Analysis of the potential of C/EBP{alpha} to increase involucrin expression in HNSCC cell lines with 5-aza-2'-deoxycytidine. A, involucrin protein expression was compared between HNSCC cell lines (SCC11B and SCC17AS) with and without 5-aza-2'-deoxycytidine using Western blot analysis with an antibody against involucrin. B, the drawing depicts the ChIP primer location surrounding the C/EBP{alpha} binding site upstream of the transcription start site of involucrin. ChIP was done using C/EBP{alpha} antibody (sc-61) on SCC22B and SCC17AS cells before and after demethylating treatment. Quantitative PCR (i.e., SYBR green) was done using involucrin promoter primers (surrounding the C/EBP{alpha} binding site) on the ChIP eluate. Values were normalized to the threshold crossing of the negative (no antibody) control. *, P < 0.0126; **, P < 0.00381. C, pictures were taken through a microscope using a digital camera [C/EBP{alpha}-overexpressing SCC22B (C/EBP{alpha}+); SCC22B (C/EBP{alpha}–)]. Quantitation of the percentage of differentiated cells was calculated for 10 frames of cells for both SCC22B and C/EBP{alpha}-overexpressing SCC22B cells. Magnification, x100 for both frames (C/EBP{alpha}-overexpressing SCC22B are much larger in size compared with untransfected SCC22B). Spindle formation was the criteria used to distinguish differentiated cells from nondifferentiated cells; thick black arrows, examples. *, P < 0.00035.

 
Furthermore, differences in cell morphology were seen through microscopic evaluation in C/EBP{alpha}-overexpressing cell lines. The C/EBP{alpha}-overexpressing SCC22B are much larger in size compared with the untransfected SCC22B cells (possibly due to accumulation of granules and/or vacuoles, which are often increased in differentiated cells; ref. 15). Using previously accepted criteria for differentiation determination (i.e., spindle formation; ref. 15), C/EBP{alpha}-overexpressing cells evidence a higher frequency of differentiated cells than the untransfected SCC22B cells (16% versus 5%, respectively; Fig. 5C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
C/EBP{alpha}, a gene involved in cellular differentiation and cell cycle control (24), has been found to exhibit tumor suppressor activity in non–small cell lung cancer (15) and AML (14). More recently, a publication revealed C/EBP{alpha} as one of 102 metastasis-predictive genes in HNSCC that exhibits distinct expression profiles (from their nonmetastatic counterpart) at the primary tumor stage (25). This is likely due to the requirement for activation of C/EBP{alpha} of differentiation genes (i.e., involucrin) in keratinocytes (23).

In our study, we have shown for the first time not only that C/EBP{alpha} overexpression provides tumor suppressor activity (and metastasis inhibition) in vitro and in vivo in HNSCC, but also that it is epigenetically down-regulated by upstream methylation. It seems surprising that we found no methylation around the transcription start site and core promoter, but this supports previous findings of C/EBP{alpha} epigenetic regulation in lung cancer (16). Therefore, it may be assumed that upstream methylation is capable of decreasing expression, but the core promoter remains unmethylated due to a requirement of basal expression for tumor cell viability (16). Deletions within chromosome 19 have not been previously discovered in HNSCC (26), and very few cases (i.e., RASSF1A and TCF21) have been documented thus far (9, 27) as having frequent co-occurrence of both DNA methylation and LOH in HNSCC, as we witnessed with C/EBP{alpha} (although there are several examples in other cancer types; refs. 28–32).

The findings of this study are of critical importance because HNSCC often has a fatal clinical course with very little chance of recovery following operation (4). Currently, there are relatively few good markers for poor-prognosis HNSCC detection. Loss of BRCA2 correlates with a significant decrease in survival time, and certain chromosomal deletions (i.e., 10q and 14q) were found to be associated with poor prognosis (33–35). Increased gene expression [i.e., NBS1, FGFR, Ki-67, HER2, matrix metalloproteinase 2 (MMP2) and MMP9, and cyclin D1] has been shown to indicate the aggressiveness of the disease (36–41), and elevated alpha B-crystallin expression has been found to be a more sensitive marker for HNSCC recurrence than the combination of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (42). However, there is still not a DNA methylation marker for poor prognosis (12). DNA methylation occurs early in carcinogenesis, making it a good early indicator (43). Consequently, the discovery of C/EBP{alpha} down-regulation in poor-prognosis patients (13), combined with our findings of epigenetic regulation of the gene, has great importance for detection of HNSCC. This suggests that there may be great promise for using DNA methylation as a biomarker in clinical screening, which has already been proposed in other cancer types (42–45). This could ultimately lead to better detection (either by immunohistochemistry or by quantitative DNA methylation analysis) and treatment of the disease, increasing the rate of survival (44). However, C/EBP{alpha} methylation in HNSCC should be validated in a large sample set before pursuing its diagnostic potential (42). Furthermore, C/EBP{alpha} methylation in other tumor types (besides lung cancer and HNSCC) is worthy of investigation and could yield increased prognostic value and therapeutic intervention for other patients with this epigenetic alteration.


   Acknowledgments
 
Grant support: National Institute of Dental and Craniofacial Research, DE13123 (C. Plass), Alumni Grants for Graduate Research and Scholarship (K. Bennett), and a postdoctoral fellowship from the Dr. Mildred Scheel Foundation for Cancer Research (B. Hackanson). C. Eng is a recipient of the Doris Duke Distinguished Clinical Scientist Award.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Gokhan Hotamisligil for the C/EBP{alpha} overexpression plasmid. The seven different established human HNSCC cell lines used in the study (SCC4, SCC5, SCC8, SCC11B, SCC17AS, SCC22B, and SCC25) were kindly provided by Dr. Thomas Carey. We thank Martin Brena and Mau-ting Lin for their thoughtful discussions.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/29/06. Revised 2/23/07. Accepted 3/15/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. St John MA, Abemayor E, Wong DT. Recent new approaches to the treatment of head and neck cancer. Anticancer Drugs 2006;17:365–75.[CrossRef][Medline]
  2. Li XM, Di B, Shang YD, et al. [Analysis of risk factors in the prediction of distant metastases of head and neck squamous cell carcinomas]. Zhonghua Er Bi Yan Hou Ke Za Zhi 2004;39:171–5.[Medline]
  3. van Oijen MG, Slootweg PJ. Oral field cancerization: carcinogen-induced independent events or micrometastatic deposits? Cancer Epidemiol Biomarkers Prev 2000;9:249–56.[Abstract/Free Full Text]
  4. Gupta T, Agarwal JP. Planned neck dissection following chemo-radiotherapy in advanced HNSCC. Int Semin Surg Oncol 2004;1:1–6.[CrossRef][Medline]
  5. Smiraglia DJ, Smith LT, Lang JC, et al. Differential targets of CpG island hypermethylation in primary and metastatic head and neck squamous cell carcinoma (HNSCC). J Med Genet 2003;40:25–33.[Abstract/Free Full Text]
  6. Marsit CJ, McClean MD, Furniss CS, et al. Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma. Int J Cancer 2006;119:1553–7.[CrossRef][Medline]
  7. Estecio MR, Youssef EM, Rahal P, et al. LHX6 is a sensitive methylation marker in head and neck carcinomas. Oncogene 2006;25:5018–26.[CrossRef][Medline]
  8. Kraunz KS, Hsiung D, McClean MD, et al. Dietary folate is associated with p16(INK4A) methylation in head and neck squamous cell carcinoma. Int J Cancer 2006;119:1761–6.[CrossRef][Medline]
  9. Smith LT, Lin M, Brena RM, et al. Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23–24 in lung and head and neck cancer. Proc Natl Acad Sci U S A 2006;103:982–7.[Abstract/Free Full Text]
  10. Crowe DL, Hacia JG, Hsieh CL, et al. Molecular pathology of head and neck cancer. Histol Histopathol 2002;17:909–14.[Medline]
  11. Marsit CJ, Liu M, Nelson HH, et al. Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival. Oncogene 2004;23:1000–4.[CrossRef][Medline]
  12. Esteves LI, Javaroni AC, Nishimoto IN, et al. DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: does positive expression predict poor prognosis in early stage head and neck carcinomas? Mol Carcinog 2005;44:102–10.[CrossRef][Medline]
  13. Roepman P, Wessels LF, Kettelarij N, et al. An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas. Nat Genet 2005;37:182–6.[CrossRef][Medline]
  14. Porse BT, Bryder D, Theilgaard-Monch K, et al. Loss of C/EBP {alpha} cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage. J Exp Med 2005;202:85–96.[Abstract/Free Full Text]
  15. Halmos B, Huettner CS, Kocher O, et al. Down-regulation and antiproliferative role of C/EBP{alpha} in lung cancer. Cancer Res 2002;62:528–34.[Abstract/Free Full Text]
  16. Tada Y, Brena RM, Hackanson B, et al. Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein {alpha} activity in lung cancer. J Natl Cancer Inst 2006;98:396–406.[Abstract/Free Full Text]
  17. Ehrich M, Nelson MR, Stanssens P, et al. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc Natl Acad Sci U S A 2005;102:15785–90.[Abstract/Free Full Text]
  18. Kim JE, Chen J. Regulation of peroxisome proliferator-activated receptor-{gamma} activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 2004;53:2748–56.[Abstract/Free Full Text]
  19. Heckman CA, Wheeler MA, Boxer LM. Regulation of Bcl-2 expression by C/EBP in t(14;18) lymphoma cells. Oncogene 2003;22:7891–9.[CrossRef][Medline]
  20. Frohling S, Schlenk RF, Stolze I, et al. CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations. J Clin Oncol 2004;22:624–33.[Abstract/Free Full Text]
  21. Timchenko N, Wilson DR, Taylor LR, et al. Autoregulation of the human C/EBP {alpha} gene by stimulation of upstream stimulatory factor binding. Mol Cell Biol 1995;15:1192–202.[Abstract]
  22. Efimova T, Eckert RL. Regulation of human involucrin promoter activity by novel protein kinase C isoforms. J Biol Chem 2000;275:1601–7.[Abstract/Free Full Text]
  23. Balasubramanian S, Agarwal C, Efimova T, et al. Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer–binding protein {alpha} (C/EBP{alpha}) DNA binding. Biochem J 2000;350 Pt 3:791–6.[CrossRef][Medline]
  24. Schrem H, Klempnauer J, Borlak J. Liver-enriched transcription factors in liver function and development: part II. The C/EBPs and D site-binding protein in cell cycle control, carcinogenesis, circadian gene regulation, liver regeneration, apoptosis, and liver-specific gene regulation. Pharmacol Rev 2004;56:291–330.[Abstract/Free Full Text]
  25. Roepman P, de Jager A, Groot Koerkamp MJ, et al. Maintenance of head and neck tumor gene expression profiles upon lymph node metastasis. Cancer Res 2006;66:11110–4.[Abstract/Free Full Text]
  26. Bockmuhl U, Wolf G, Schmidt S, et al. Genomic alterations associated with malignancy in head and neck cancer. Head Neck 1998;20:145–51.[CrossRef][Medline]
  27. Hogg RP, Honorio S, Martinez A, et al. Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma. Eur J Cancer 2002;38:1585–92.[CrossRef][Medline]
  28. McKie AB, Douglas DA, Olijslagers S, et al. Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer. Oncogene 2005;24:2166–74.[CrossRef][Medline]
  29. Chopin V, Leprince D. [Chromosome arm 17p13.3: could HIC1 be the one?]. Med Sci (Paris) 2006;22:54–61.
  30. Woo IS, Park MJ, Choi SW, et al. Loss of estrogen receptor-{alpha} expression is associated with hypermethylation near its ATG start codon in gastric cancer cell lines. Oncol Rep 2004;11:617–22.[Medline]
  31. Nicoll G, Crichton DN, McDowell HE, et al. Expression of the hypermethylated in cancer gene (HIC-1) is associated with good outcome in human breast cancer. Br J Cancer 2001;85:1878–82.[CrossRef][Medline]
  32. Kuramochi M, Fukuhara H, Nobukuni T, et al. TSLC1 is a tumor-suppressor gene in human non–small-cell lung cancer. Nat Genet 2001;27:427–30.[CrossRef][Medline]
  33. Sabbir MG, Roy A, Mandal S, et al. Deletion mapping of chromosome 13q in head and neck squamous cell carcinoma in Indian patients: correlation with prognosis of the tumour. Int J Exp Pathol 2006;87:151–61.[CrossRef][Medline]
  34. Gasparotto D, Vukosavljevic T, Piccinin S, et al. Loss of heterozygosity at 10q in tumors of the upper respiratory tract is associated with poor prognosis. Int J Cancer 1999;84:432–6.[CrossRef][Medline]
  35. Lee DJ, Koch WM, Yoo G, et al. Impact of chromosome 14q loss on survival in primary head and neck squamous cell carcinoma. Clin Cancer Res 1997;3:501–5.[Abstract]
  36. Yang MH, Chiang WC, Chou TY, et al. Increased NBS1 expression is a marker of aggressive head and neck cancer and overexpression of NBS1 contributes to transformation. Clin Cancer Res 2006;12:507–15.[Abstract/Free Full Text]
  37. Silva SD, Agostini M, Nishimoto IN, et al. Expression of fatty acid synthase, ErbB2 and Ki-67 in head and neck squamous cell carcinoma. A clinicopathological study. Oral Oncol 2004;40:688–96.[CrossRef][Medline]
  38. Streit S, Bange J, Fichtner A, et al. Involvement of the FGFR4 Arg388 allele in head and neck squamous cell carcinoma. Int J Cancer 2004;111:213–7.[CrossRef][Medline]
  39. Masuda M, Suzui M, Lim JT, et al. Epigallocatechin-3-gallate inhibits activation of HER-2/neu and downstream signaling pathways in human head and neck and breast carcinoma cells. Clin Cancer Res 2003;9:3486–91.[Abstract/Free Full Text]
  40. Xu YP, Zhao XQ, Sommer K, et al. Correlation of matrix metalloproteinase-2, -9, tissue inhibitor-1 of matrix metalloproteinase and CD44 variant 6 in head and neck cancer metastasis. J Zhejiang Univ Sci 2003;4:491–501.[Medline]
  41. Namazie A, Alavi S, Olopade OI, et al. Cyclin D1 amplification and p16(MTS1/CDK4I) deletion correlate with poor prognosis in head and neck tumors. Laryngoscope 2002;112:472–81.[CrossRef][Medline]
  42. Chin D, Boyle GM, Williams RM, et al. Alpha B-crystallin, a new independent marker for poor prognosis in head and neck cancer. Laryngoscope 2005;115:1239–42.[CrossRef][Medline]
  43. Dhawan D, Hamdy FC, Rehman I, et al. Evidence for the early onset of aberrant promoter methylation in urothelial carcinoma. J Pathology 2006;209:336–43.[CrossRef][Medline]
  44. Matsubayashi H, Canto M, Sato N, et al. DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease. Cancer Res 2006;66:1208–17.[Abstract/Free Full Text]
  45. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148–54.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J.-S. Chung, T. Yudate, M. Tomihari, H. Akiyoshi, P. D. Cruz Jr., and K. Ariizumi
Binding of DC-HIL to Dermatophytic Fungi Induces Tyrosine Phosphorylation and Potentiates Antigen Presenting Cell Function
J. Immunol., October 15, 2009; 183(8): 5190 - 5198.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. E. Figueroa, B. J. Wouters, L. Skrabanek, J. Glass, Y. Li, C. A. J. Erpelinck-Verschueren, A. W. Langerak, B. Lowenberg, M. Fazzari, J. M. Greally, et al.
Genome-wide epigenetic analysis delineates a biologically distinct immature acute leukemia with myeloid/T-lymphoid features
Blood, March 19, 2009; 113(12): 2795 - 2804.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
S. Koschmieder, B. Halmos, E. Levantini, and D. G. Tenen
Dysregulation of the C/EBP{alpha} Differentiation Pathway in Human Cancer
J. Clin. Oncol., February 1, 2009; 27(4): 619 - 628.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
B. Hackanson, K. L. Bennett, R. M. Brena, J. Jiang, R. Claus, S.-S. Chen, N. Blagitko-Dorfs, K. Maharry, S. P. Whitman, T. D. Schmittgen, et al.
Epigenetic Modification of CCAAT/Enhancer Binding Protein {alpha} Expression in Acute Myeloid Leukemia
Cancer Res., May 1, 2008; 68(9): 3142 - 3151.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. S. Hasemann, I. Damgaard, M. B. Schuster, K. Theilgaard-Monch, A. B. Sorensen, A. Mrsic, T. Krugers, B. Ylstra, F. S. Pedersen, C. Nerlov, et al.
Mutation of C/EBP{alpha} predisposes to the development of myeloid leukemia in a retroviral insertional mutagenesis screen
Blood, April 15, 2008; 111(8): 4309 - 4321.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bennett, K. L.
Right arrow Articles by Plass, C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Bennett, K. L.
Right arrow Articles by Plass, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online