Inducible nitric oxide synthase, nitrotyrosine and p53 mutations in the molecular pathogenesis of Barrett's esophagus and esophageal adenocarcinoma

Abstract

Nitric oxide (NO) has been implicated as a potential causative factor for endogenous p53 mutations in gastrointestinal malignancy. To investigate the role of NO in esophageal adenocarcinoma (EADC), we studied patterns of p53 mutations, expression of inducible nitric oxide synthase (iNOS) and the tissue accumulation of nitrotyrosine (NTS), a stable reaction product of NO and a marker for cellular protein damage, in human premalignant and malignant esophageal epithelia. Tissues were obtained from patients with gastroesophageal reflux disease (GERD)-induced esophagitis (n = 76), Barrett's esophagus (BE; n = 119) and primary EADC (n = 54). DNA sequencing was used to characterize p53 mutations, RT-PCR to study iNOS mRNA expression, and immunohistochemistry to study NTS. Relative to self-matched normal epithelia, a progressive increase in iNOS mRNA expression was seen in GERD (30%; 23/76), BE (48%; 57/119), and EADC (63%; 34/54) tissues (P< 0.001). Among patients with EADC, elevated levels of NTS immunoreactivity were more frequent in tumors with p53 mutations (11/21; 52%) compared with tumors with wild-type p53 (9/33; 27%; P = 0.063), and specifically in tumors with p53 mutations at CpG dinucleotides (10/12; 83%) compared with non-CpG p53 mutations (1/9; 11%; P = 0.008). The increasing frequency of iNOS (mRNA) overexpression in GERD, BE and EADC supports the hypothesis that an active inflammatory process, most likely a consequence of GERD, underlies molecular progression to EADC. The highly significant association between NTS, reflecting chronic NO-induced cellular protein damage, and endogenous p53 mutations at CpG dinucleotides, provides further evidence for a molecular link between chronic inflammation and esophageal malignancy.

TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use

“…The 5 0 methylcytosine (5mC) is less stable than cytosine and undergoes spontaneous deamination into thymine at a rate five times higher than the unmethylated base. This process is enhanced by oxygen and nitrogen radicals, leading to a higher load of CpG transitions in cancers arising from inflammatory precursors such as Barrett's mucosa or ulcerative colitis (Schmutte et al 1996;Ambs et al 1999;Vaninetti et al 2008). Among the 22 CpG of the DNA-binding domain (DBD), three hotspot codons (175, 248, and 273) Mutation pattern/24785 mutations (196, 213, 245, 282, and 306) account for 26% of these mutations.…”

Prognostic value of p53 mutations in oesophageal adenocarcinoma: final results of a 15-year prospective study

“…Polymerase chain reaction (PCR)-based molecular assays and immunohistochemical techniques were used to characterise p53 mutations and protein accumulation, respectively, as previously reported [9,10,13]. Briefly, genomic DNA was extracted from banked oesophageal tissues (tumour and matched normal epithelium as an internal control), and p53 exons 4-10 amplified by PCR.…”

“…Strict clinicopathologic criteria [8][9][10] were used to define primary EADCs (Siewert classification Type I). As resected oesophageal tissues were used for molecular studies [9,10,13], additional sections of the primary tumour (comprising >80% malignant cells, with minimal necrosis), each with matched histologically normal oesophageal epithelia (obtained from a distant site, adjacent to the proximal resection margin) were snap-frozen in liquid nitrogen at the time of resection, and stored in an oesophageal tumour bank at À80 8C. Approval for banking surgically resected oesophageal tissues for molecular studies was obtained from review boards for health science research at the Universities of Western Ontario (London) and Toronto (Ontario, Canada) and respective Departments of Pathology.…”

Barrett’s oesophagus: an ideal model to study cancer genetics

“…Sustained induction of the inducible form of NOS (iNOS) in chronic inXammation may be mutagenic, through NO-mediated DNA damage or hindrance to DNA repair (Lala and Chakraborty 2001). Levels of iNOS increase in oesophageal tissue samples of oesophagitis and Barrett's compared to normal specimens (Vaninetti et al 2008). In the context of GORD NO can also be generated by the reduction of salivary nitrite by acid (Suzuki et al 2005) or by reduction of nitrosating species present in the gastric acid environment by dietary ascorbic acid (Iijima et al 2003;Combet et al 2007).…”