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Ann Thorac Surg 2007;83:433-440
© 2007 The Society of Thoracic Surgeons
a Cardiothoracic-Renal Molecular Research Program, Johns Hopkins University School of Medicine, Baltimore, Maryland
b Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
c Department of Surgery, Johns Hopkins Bayview Medical Center, Baltimore, Maryland
Accepted for publication June 28, 2006.
* Address correspondence to Dr Wei, Johns Hopkins University School of Medicine, 600 N. Wolfe St, Blalock 1206, Baltimore, MD 21205 (Email: cmwei{at}jhmi.edu).
Presented at the Forty-first Annual Meeting of The Society of Thoracic Surgeons, Tampa, FL, Jan 24–26, 2005.
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Abstract |
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METHODS: Reflux was promoted in male Sprague-Dawley rats by duodenoesophageal anastomosis (8 weeks) without gastric bypass. MNAN treatment (25 mg/kg per week intraperitoneally for four doses) commenced at 10 weeks age. Ten animals served as controls. Quantification of 8-oxoG was performed by using immunohistochemistry, and MYH was analyzed by Western blot. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL), cytochrome C, and caspase.
RESULTS: Tumors (adenocarcinoma) developed in 15 (50%) of 30 animals with reflux alone; this increased to 26 (86.6%) of 30 when reflux was combined with MNAN treatment, with tumor histology consistent with adenosquamous and squamous cell cancer. DNA damage, as reflected by positive 8-oxoG staining in reflux groups, was significantly increased compared with control (p < 0.01), and this was maximal in tissues with malignant transformation. Protein levels of the DNA repair enzyme MYH were significantly less in tissues subjected to reflux compared with controls (p < 0.05). TUNEL, cytochrome C, and caspase positivity confirmed increased apoptosis in cancer lesions.
CONCLUSIONS: Gastroduodenal reflux leads to increased DNA damage and downregulation of the DNA mismatch repair pathway. This pathway has an important role in esophageal carcinogenesis in rats.
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Introduction |
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Long-term excessive production of reactive oxygen species has an important role in the modulation of GERD-associated esophageal mucosal damage [5, 6]. Reactive oxygen species, including superoxide radical, hydrogen peroxide (H2O2), hydroxyl radical, and the peroxynitrite radical, are critical in the pathogenesis of GERD-mediated esophageal mucosal damage and subsequent malignant transformation [6, 7]. A recent report suggests the mutagenic potential of duodenoesophageal reflux in an experimental model of esophageal cancer with deleterious base transitions [8].
One of the most stable, deleterious products of oxidative DNA damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG) [9]. Unrepaired 8-oxoG lesions in DNA can lead to A/8-oxoG mismatches during DNA replication, and result in G:C to T:A mutations [10, 11]. The Mut-Y homologue (MYH) gene removes the adenine misincorporated opposite 8-oxoG, G, or C after DNA replication [12]. Inherited biallelic mutations in the MYH gene have been shown to be responsible for autosomal recessive syndrome of adenomatous colorectal polyposis and a subsequent high risk of developing colorectal cancer [13].
We conducted this study to investigate the role of DNA damage and its repairing mechanisms, particularly MYH, in esophageal cancer. We hypothesize that the malignant transformation in the esophageal mucosa is a result of chronic disparity between DNA injury and repair induced owing to GERD. We aimed to study the DNA damage and repair ability by MYH in adenocarcinoma and squamous cell cancer, the two histologic variants of esophageal cancer. We report our investigation of the role of MYH in esophageal carcinogenesis in a rat model of GERD [14].
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Material and Methods |
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The rats were fasted overnight, but were allowed water ad libitum until the surgery. Briefly, the esophagus was mobilized by preserving the vagus nerves and anastomosed side-to-side to the first part of duodenum immediately distal to the pyloric sphincter. The anastomotic sutures were carefully placed a millimeter from the edge of the pyloric sphincter to achieve duodenal and gastric reflux simultaneously in the lower esophagus, before neutralization of the gastric contents could take place in the duodenum. Care was taken to preserve the pyloric sphincter and cardiac sphincter to maintain the integrity of the stomach. The detailed procedure is described elsewhere [14].
Once the animals awoke, they were allowed water ad libitum, and feeding recommenced the next day. Animals received appropriate analgesia during the perioperative and postoperative period.
Another group that underwent operation received methyl-n-amyl nitrosamine (MNAN), injected intraperitoneally (25 mg/kg body weight; 50% LD50), for four doses administered weekly for 4 weeks beginning 2 weeks after recovery from surgery. The MNAN was supplied by Dr Sidney Mirvish, University of Nebraska. The control group was 10 animals that did not undergo surgery or receive MNAN administration.
The experiment concluded 40 weeks postoperatively. Animals received humane care in compliance with the "Guide for Care and Use of Laboratory Animals," published by the National Research Council (National Academy Press, 1996). The Institutional Animal Care and Use Committee (IACUC) approved all the procedures in this study.
Tumor Harvesting
Animals were anesthetized as described before. Abdominal and thoracic viscera were exposed through a midline incision, and the tumors were assessed for size and extent. The esophagus was mobilized from the neck to the anastomosis. The tumors were identified and carefully dissected away from the liver and adjoining structures. The esophagus was opened longitudinally to expose the lumen, and half of the esophagus was preserved in 10% formalin for histologic examination and another half in liquid nitrogen for immunoblot examination.
After confirmation of the histology, two blinded histologists determined DNA damage, and performed immunohistochemistry, immunoblot examination, and terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) assay estimation on the confirmed adenocarcinoma, squamous cell cancer, and control (normal) esophageal samples. The histologic gradations are described in detail in the Appendix.
Determination of DNA Damage
Formalin-fixed, paraffin-embedded sections were evaluated for 8-oxoG with anti-8 oxoG antibody (Trevigen, Gaithersburg, MD) as previously reported [15]. Briefly, the slides were fixed and washed with phosphate buffered saline (PBS; pH 7.4) and incubated for 40 minutes at 37°C. The DNA was denatured by soaking the slides in 4 sodium hydrochloride for 7 minutes. After incubation with 50 mmol/L Tris base for 5 minutes at room temperature and two washings with PBS, 10% fetal bovine serum was added for 1 hour at room temperature to block nonspecific staining sites.
Next, slides were incubated with 3% H2O2 for 30 minutes at room temperature to block endogenous peroxidase. Thereafter, slides were incubated with primary anti-8-oxoG monoclonal antibody (diluted 1:100 in 10 mmol/L Tris-HCl, pH 7.5, 10% serum) overnight at 4°C, rinsed twice with PBS, and incubated with secondary antimouse antibody (1:100) conjugated with 20 µg/mL streptavidin-horseradish peroxidase in 1x PBS for 1 hour at room temperature.
After staining with diamino benzamide tetrahydrochloride (DAB, DAKO Corp, Carpinteria, CA) and counterstaining with methyl green, slides were examined under light microscope. The percentage of cells staining positive for 8-oxoG was quantified as follows: (positive cells/total cells) x 100 = percent 8-oxoG-positive cells.
Evaluation of Mut-Y Homologue Expression
MYH expression was assessed by two different methods, immunocytochemistry and immunoblot analysis, in the same animals studied for 8-oxoG staining.
Immunohistochemistry
Immunohistochemical staining was performed as previously described [15]. Briefly, after slides were deparaffinized, sections were rinsed in 0.1 mol/L PBS for 20 minutes and blocked in 2% normal horse serum for 2 hours. Then sections were incubated with primary MYH antibody (1:100) in 10% normal horse serum or 10% normal rabbit serum and 0.3% Triton-X 100 for 20 hours at 4°C. After endogenous peroxidase activity was quenched by exposure of the slides to 0.3% H2O2 and 10% methanol for 20 minutes, the slides were washed in PBS and incubated with secondary antibody conjugated to horse radish peroxidase (Amersham Biosciences Limited, Buckinghamshire, UK). The final reaction was achieved by incubating the sections with freshly prepared reagent containing 3-amino-9-ethylcarbazole (Sigma-Aldrich, St Louis, MO) dissolved in dimethyl formamide and sodium acetate. The sections were counterstained with hematoxylin, mounted, and reviewed with an Olympus microscope (Tokyo, Japan).
Two trained independent observers reviewed each section. Sample identities were concealed during scoring, and at least three samples were scored per group. Results are expressed as mean ± standard error of the mean for each group. For each sample, MYH expression was evaluated by scoring the percentage of positive staining on the entire section. According to this scoring system, 0, no staining; 1, minimal staining (<10% section positive); 2, mild staining (10% to 30% section positive); 3, (31% to 50% positive); and 4, strong staining (>50% section positive). The specificity of positive staining was further confirmed by substitution of normal rabbit serum for the primary antiserum.
Immunoblot Analysis
Western blot analysis was performed as described previously [15]. Briefly, tissue samples were homogenized in a lysis buffer (0.1 mol/L sodium chloride; 0.01 mol/L Tri-HCl, pH 7.5, 1 mmol/L ethylenediaminetetraacetic acid; and 1 µg/mL aprotinin), and then the homogenates were centrifuged at 7000g for 15 minutes at 4°C. Supernatants were used as protein samples. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis was performed in a 10% polyacrylamide gel. Protein samples were boiled at 100°C in 2.5% SDS and 5% mercaptoethanol. Lysates equivalent to 20 µg of protein from each sample were run on the gel for 90 minutes at 20 mA, together with a size marker (Invitrogen, Carlsbad, CA). The electrophoresis running buffer contained 25 mmol Tris base, 250 mmol/L glycine, and 0.1% SDS. Proteins on the gel were transferred to a nitrocellulose membrane with a transfer buffer that consisted of 48 mmol/L Tris base, 39 mmol/L glycine, 0.4% SDS, and 20% methanol.
After transfer, membranes were placed in 1% powdered milk in PBS to block nonspecific binding. After reacting with the primary and secondary antibodies, the membrane was subjected to the Enhanced Chemiluminescence analysis system (Amersham Biosciences). Polyclonal antibody against MYH was obtained from Novus Biological (Littleton, CO). Monoclonal antibody against actin (Ab-6, Oncogene Research Products, Cambridge, MA) was used to control for differences in protein loading. To ascertain specific binding of the anti-MYH antibody, another membrane was studied without this primary antibody. Densitometric analysis was accomplished by scanning x ray images of the membranes (Fluorine-S Multimager, Bio-Rad, Hercules, CA). The resolution of the images was 100 pixels/inch. A densitometry image analysis software package (Bio-Rad) was used.
Evaluation of Cell Death
To quantify the relative number of cells with DNA fragmentation, TUNEL assay was performed in esophageal sections from each rat according to our previously reported method [15] using an ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Briefly, after the sections were deparaffinized, tissue nuclei were stripped of proteins by incubation with 20 µg/mL proteinase K for 10 minutes. After treatment with 0.3% H2O2 in distilled water for 5 minutes, the sections were incubated with terminal deoxynucleotidyl transferase buffer (TdT, Boehringer Mannheim, Indianapolis, IN), 30 mM Tris (pH 7.2), 140 mM sodium cacodylate, and 1 mM cobalt chloride containing TdT enzyme (0.5 U/mL, Boehringer Mannheim) and biotin-16-2'-deoxyuridine 5'-triphosphate (0.04 mM; Boehringer Mannheim) containing 30 mmol/L cobalt chloride in a humidified chamber at 37°C for 120 minutes. The reaction was terminated by incubating with 300 mM sodium chloride and 30 mM sodium citrate for 15 minutes at 25°C.
After washing with 50 mM Tris-HCl (pH 7.7), sections were stained with diaminobenzidine (DAB)/H2O2 solution and counterstained with hematoxylin. After three washes in Tris-HCl (pH 7.7) sections were dehydrated in ascending ethanol series, immersed in xylene, and coverslips were mounted with Permount (Biomeda, Foster City, CA).
To determine the percentage of dead cells, TUNEL-positive and TUNEL-negative cells were counted. Results are expressed as number of TUNEL-positive cells/total number of cells per field x100. In addition, estimation of cytochrome C and caspase was done to estimate the DNA damage according to standard manufacturer’s protocol.
Statistical Analysis
Results of all quantitative studies are expressed as mean ± SE. Statistical comparisons within each group were performed with analysis of variance for repeated measures, followed by the Fisher least-significant difference test of repeated measures when appropriate. Comparisons between groups were performed with factorial analysis of variance, followed by the Fisher least-significant difference test of repeated measures. Statistical significance was accepted at two-tailed p < 0.05.
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Results |
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We studied the DNA damage and repair in two histologic variants of esophageal cancer, ADC and SCC. We found positive 8-oxoG staining in both, but no positive staining was found in normal rat esophagus (Fig 2A). Quantitative analysis of 8-oxoG staining (Fig 2B) indicated that the 8-oxoG level was significantly higher in ADC and SCC compared with normal rat esophagus (<1% versus 45% to 50%; p < 0.01).
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Comment |
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Genomic instability can be detrimental owing to the interaction between potential mutagens and defects in DNA repair and can lead to malignant transformation by selective clonal expansion. We used a novel model of reflux-induced esophageal tumors, which closely mimics human GERD-related esophageal cancer [14]. Histologic similarities between the reflux-induced rat model and human esophageal cancer have been previously shown [16].
We have demonstrated that DNA damage, as estimated by 8-oxoG levels, was significantly increased in both ADC and SCC. However, the expression and activity of the DNA mismatch repair enzyme MYH was significantly depressed in ADC and SCC, implying that an exhausted repair system together with increased DNA damage can have a significant role in malignant progression. Understanding these pathways can offer novel therapeutic avenues to reverse or halt these processes.
GERD has been shown to increase the production of oxygen free radicals in esophageal mucosa in humans [17, 18]. The mutagenic potential of GERD contents in a reflux-induced rat esophagitis model has been demonstrated recently [8]. A human study investigating the role of oxidative stress in GERD-related esophageal cancer progression sequence reported the formation of DNA adducts, increased myeloperoxidase-related oxidative stress, and decreased antioxidant capacity together with a negative correlation between glutathione content and DNA adducts [7]. Results from an animal study suggested a possible protective role of antioxidants in reflux esophagitis [19].
DNA strand breaks can take place owing to reactive oxygen species injury. Amongst the different oxidative stress–induced DNA damage products, 8-oxoG is the most stable and deleterious adduct. This can result in mismatch base pairs by polymerases during the DNA replication phase. If this lesion is not repaired properly, it can lead to G:C to T:A transversion. MYH plays an important role in the repairing of this lesion. Mut-Y, Mut-M, and Mut-T are the family of DNA repair enzymes involved in defending against the mutagenic effects of 8-oxoG lesions in Escherichia coli [20]. The DNA repair pathways that protect cells from the mutagenic effects of 8-oxoG are highly conserved. Human Mut-Y glycosylase homologue (hMYH) shares high sequence homology and similar mechanisms with the E coli Mut-Y protein [21]. The MYH protein protects the cell from the mutagenic effects of 8-oxoG.
Mutations and alterations in MYH have been implicated in the autosomal recessive nonfamilial colonic polyposis and have been shown to increase the subsequent risk of colorectal cancers [13]. In a study examining 95 sporadic gastric cancers, two cancers were found to have biallelic mutations of the MYH gene, with somatic missense of one and loss of the remaining allele in another. The two cancers were advanced intestinal-type gastric cancers with lymph node metastasis. Four of 23 cases showed allele loss at the MYH locus, suggesting that somatic mutations in the MYH gene may lead to some of these cancers [22]. Mice deficient in base repair enzymes have been shown to develop various cancers, including lung cancers in more than 60% of animals [23]. Age-associated 8-oxoG lesions and subsequent development of cancers have been demonstrated in MYH knockout mice [24]. This suggests a prominent role for base repair enzymes such as MYH in preventing tumorigenesis. Similar human data on the role of mutations in MYH and subsequent esophageal cancer risk is currently lacking.
The present investigation raises an important issue regarding the role of MYH in esophageal carcinogenesis. We have demonstrated that the MYH activity and concentration is significantly exhausted in tissues with malignant changes. Further studies are warranted to elucidate the precise role played by mutations in MYH and subsequent esophageal cancer risk in patients with GERD.
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Appendix |
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Precancerous Squamous Lesions: This term was chosen to describe a common finding of basal cell hyperplasia, papillary elongation, and hyperkeratosis. A squamous papilloma was diagnosed when there was a localized papillary proliferation of benign squamous epithelium.
Columnar Metaplasia (Barrett Esophagus): Defined as the presence of unequivocal columnar epithelium (intestinal type goblet cells) above the anastomosis confirmed by periodic acid Schiff and Alcian blue staining.
Dysplasia and Malignant Transformation: Diagnosis of dysplasia was based on the abnormal cell polarity, maturation, nuclear atypia, and mitotic figures. Tumors were defined on the basis of malignant cells infiltrating the mucosa or basement membrane of the oesophageal wall. Depth of invasion was classed as confined to the mucosa or submucosa, into the muscularis propria, and through the muscularis propria. Malignant transformation was graded as carcinoma in situ, squamous cell carcinoma in tumors with a pure squamous morphology; tumors with definite adenocarcinomatous elements were classified as adenocarcinoma. Mucin secretion was confirmed on mucicarmine staining.
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Discussion |
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DR BONDE: Thank you for your questions. Regarding your first question, whether we looked at the expression as per the sequential progression, for the purpose of this study, we killed the animals at 32 weeks when they already developed cancer because we wanted to answer the question whether MYH expression and the DNA damage is increasing tumors or not. Regarding your second question, why we had most squamous cell cancers in the second group, the second group had the MNAN administration, and it is known that whenever you have methyl-n-amyl nitrosamine administration, you have a higher incidence of squamous cell cancers. Regarding the third question about whether we are doing further studies using mRNA, that is our current investigation and focus and that is what we plan to elucidate in the next few months.
DR M. BLAIR MARSHALL (Washington, DC): I thought that was an excellent study as well. Have you looked at these pathways in human tumor specimens to determine the potential role of this mechanism development of cancer in our patients?
DR BONDE: Well, that’s a very good question. The reason why we didn’t look into human tumors straightaway was twofold. One is that there are several factors involved in terms of the collection of history and everything to say whether these are reflux-induced tumors or whether they are reflux-related tumors or not. That was the first question. The second thing was that in our model, we can control these factors and investigate this further. However, we are planning to look at particularly polymorphism in this particular gene, MYH, which has been shown in lung cancer as well as in colorectal cancer to increase the risk of developing subsequent cancer. Naturally, the results of those studies will take some time to come. So that is the reason why we used the animal model.
DR DAVID W. JOHNSTONE (Lebanon, NH): Is this experimental model one of biliary reflux model, acid reflux, or a combination?
DR BONDE: This is a model that causes reflux of both acid as well as bile. The anastomosis is made between the first part of the duodenum and the lower part of the esophagus, but care is taken that the incision on the duodenum goes right up to the pylorus, so that when the stitch is taken, the pylorus is almost all refluxing into the lower part of the esophagus, unlike other models where the anastomosis is made much higher, and the duodenal contents actually neutralize most of the gastric secretions by the time they reflux into the esophagus.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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T and AC substitutions J Biol Chem 1992;267:166-172.T:A transversions induced by a single 8-oxoguanine residue in single-stranded DNA Mol Gen Genet 1993;239:72-76.[Medline]
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