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Oxidative Stress and p53 Mutations in the Carcinogenesis of Iron Overload-Associated Hepatocellular Carcinoma

Aizen J. Marrogi, Mohammed A. Khan, Hilda E. van Gijssel, Judith A. Welsh, Haress Rahim, Anthony J. Demetris, Kris V. Kowdley, S. Perwez Hussain, Jagdish Nair, Helmut Bartsch, Nadir Okby, Miriam C. Poirier, Kumal G. Ishak, Curtis C. Harris

Affiliations of authors: A. J. Marrogi, M. A. Khan, J. A. Welsh, H. Rahim, S. P. Hussain, C. C. Harris (Laboratory of Human Carcinogenesis, Center for Cancer Research), H. E. van Gijssel, M. C. Poirier (Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research), National Cancer Institute, Bethesda, MD; A. J. Demetris, Department of Pathology and Laboratory Medicine, University of Pittsburgh, PA; K. W. Kowdley, Department of Surgery, University of Washington, Seattle; J. Nair, H. Bartsch, German Cancer Center, Heidelberg, Germany; N. Okby, K. G. Ishak, Department of Hepatic and Gastrointestinal Pathology, The Armed Forces Institute of Pathology, Washington, DC.

Correspondence to: Curtis C. Harris, M.D., National Institutes of Health, Bldg. 37, Rm. 2C05, MSC 4255, Bethesda, MD 20892–4255 (e-mail: Curtis_Harris{at}nih.gov).

The incidence of hepatocellular carcinoma (HCC) in individuals with hereditary hemochromatosis is 200 times greater than in the general population (1). This increased risk of HCC may be the result of deregulation of oxidation reduction and generation of reactive oxygen species from free iron, directly through the Fenton reaction and indirectly through the acceleration of lipid peroxidation [reviewed in (2)]. In an iron–nitrilotriacetic acid rat model of hemochromatosis, renal samples showed an increase in the levels of several reactive intermediates, including 4-hydroxy-2-nonenal and malondialdehyde (3,4), both of which are known to be cytotoxic and genotoxic (5,6). These increases were accompanied by decreased availability of systems that protect against oxidation by iron, such as vitamin E levels, the glutathione system, thiol-specific antioxidants, and superoxide dismutase (7,8). In addition, excess iron provides a strong growth-promotion advantage in human hepatoma cell lines and chemically induces carcinomas in experimental animals (9,10)

Previously, we have reported higher frequencies of p53 mutations, including G : C to T : A transversions at codon 249 and C : G to A : T and C : G to T : A changes at codon 250 in liver tissue samples from cancer-free patients with either hemochromatosis or Wilson's disease (11). We present here a detailed analysis of 14 cases of hemochromatosis-associated HCC (Fig. 1Go, A), including the p53 mutation spectrum, Prussian blue stain for iron (Fig. 1Go, B), and immunohistochemical analysis of p53, nitric oxide synthase-2 (NOS-2), and cyclooxygenase-2 (COX-2). The study protocol was submitted to the human subjects committee at our institution but was exempted, because the tissues were obtained in the course of patients' treatment and without the knowledge of patients' identities. Histologically, all tumors, except three, were well differentiated. Nuclear p53 overexpression was observed in eight of 14 HCC cases (Fig. 1Go, C) with varying intensity, including two (+1), two (+2), three (+3), and one (+4), according to the following scoring scheme: less than 10% nuclear reactivity = 0, greater than 11% to less than 25% = +1, greater than 25% to less than 50% = +2, greater than 50% to less than 90% = +3, and greater than 90% = +4 (12,13). Cytoplasmic NOS-2 immunoreactivity was seen in one HCC and in eight adjacent non-neoplastic regenerative nodules (Fig. 1Go, D). COX-2 overexpression was not seen in these cases of HCC.



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Fig. 1. Histopathology and immunohistochemistry (IHC) stains of human hemochromatosis-associated hepatocellular carcinoma (HCC). A) Hematoxylin–eosin (H&E) section representative of HCC with infiltrating neoplastic hepatocytes invading the adjacent regenerative nodules. B) Prussian blue stain for iron demonstrating intracellular ferrous iron deposits in hepatocytes. In addition, extracellular iron pigmentation deposits were detected. C) Nuclear p53 overexpression in a case of HCC. D) Anti-nitric oxide synthase-2 (NOS-2) immunoreactivity in regenerative nodule of HCC-associated cirrhosis in cytoplasmic pattern. In addition to hepatocytes, NOS-2 overexpression also was seen in some mononuclear inflammatory cells in the areas of piecemeal necrosis. Original magnification x20, AD. Methods: Fourteen cases of hemochromatosis-associated HCC were identified. The diagnosis of hemochromatosis was made on each patient by use of the clinical criteria of serum transferrin–iron saturation, elevated serum ferritin levels, and confirmatory hepatic iron overload of greater than 2 mol iron/g dry weight. In addition to H&E light microscopy examination, 4-µm tissue sections were cut from paraffin blocks and processed for IHC as described previously (22,34). Anti-NOS-2, anti-cyclooxygenase-2 (COX-2) (Transduction Laboratories, Lexington, KY), and anti-p53 monoclonal antibody clone D07 (Dako Corp., Santa Barbara, CA) were used at dilutions of 1 : 125, 1 : 25, and 1 : 50, respectively. Immunoreactivity for p53, NOS-2, and COX-2 was scored as reported previously (12,13) and as follows: less than 10% nuclear reactivity = 0, greater than 11% to less than 25% = +1, greater than 25% to less than 50% = +2, greater than 50% to less than 90% = +3, and greater than 90% = +4.

 
p53 mutations in nine of 14 HCCs were identified by p53 microarray analysis (14) and confirmed by automated DNA sequencing. Two possible hotspots were identified in exon 7 (codon 275, three tumors), with A : T to C : G transversions, and in exon 8 (codon 298, two cases), with G : C to C : G transversions. Overall, G : C to C : G transversions were present in four (44%) of nine cases, and three (33%) were A : T to C : G transversions. The two other mutations included a G : C to A : T transition and a G : C to T : A transversion (Fig. 2Go, A). Substantial differences were apparent in p53 mutation spectra from these HCC when compared with those reported by Vautier et al. (15). Although the prevalence of p53 mutations in both studies was similar (71% versus 64%), 60% of the tumors displayed A : T to G : C transitions, and 40% showed A : T to T : A transversions in the report by Vautier et al. (15) (Fig. 2Go, B). Some of the differences may be attributable to possible differences in the prevalence of other contributing factors between the two studies, such as viral hepatitis and alcoholic liver disease. In addition, both the incidence and spectra are vastly different from HCC of other causes (16) (compare panels A–C with panel D, Fig. 2Go). These differences suggest that multiple underlying mechanisms can lead to HCC, and that these mechanisms are different in hemochromatosis-associated HCC and HCC associated with other etiologic factors, such as aflatoxin B1 and viral hepatitis (17). Furthermore, although we have previously reported higher frequencies of G : C to T : A transversions at codon 249 and C : G to A : T transversions in codon 250 in non-neoplastic liver samples from patients with hemochromatosis and Wilson's disease (11), we were not able to detect similar p53 genetic alterations in neoplastic samples. It is possible that mutational spectra in p53 similar to those observed here would be detectable in non-neoplastic specimens from the same patients. However, mutational load assays could not be performed because no fresh-frozen tissues were available. On the other hand, if this were the case and similar p53 mutational spectra were also detected in non-neoplastic tissues in codons 275 and 298, it is possible that these mutations could provide cellular growth advantage over mutations elsewhere in the p53 gene. In vitro studies have shown "gain of function" such as transactivation, allowing a cellular growth advantage by certain p53 mutations, such as His 273 (17), and centrosome amplification followed by multipolar mitotic cell division, as seen with mouse His 172 (equivalent to human His 175) (18). Increased cellular concentrations of iron can enhance growth as well (9,10).



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Fig. 2. p53 mutation spectra in hepatocellular carcinoma (HCC). A) p53 mutation spectrum in 14 cases of hemochromatosis-associated HCC. Although the incidence of p53 mutations was similar in our case series and in those cases reported by Vautier et al. (15) (64% versus 71%), marked differences were observed. Forty-five percent of the samples exhibited G : C to C : G transversions and another 33% showed A : T to C : G transitions compared with these data (B), whereas 60% of the tumors (a total of 10 mutations among 14 samples) showed A : T to G : C and another 40% showed A : T to T : A. Methods: Slides were deparaffinized with xylene and ethanol, microdissected, and digested at 37 °C in a solution of sodium dodecyl sulfate/Proteinase K for several days until tissues were dissolved completely. DNA extraction was performed by use of the phenol–chloroform method as described elsewhere (35). Then, the p53 status was established by use of sample DNA and p53 gene chip methodology according to the manufacturer's protocol (Affymetrix, Santa Barbara, CA). Exons 2–11 of the p53 gene were amplified by use of a multiplex polymerase chain reaction, fragmented and labeled with fluorescein adenosine monophosphate tag, and hybridized onto a GeneChipTM with 2 nM control oligonucleotide, supplied by the manufacturer, for 30 minutes at 45 °C (Affymetrix). The resultant chips were scanned on a Hewlett-Packard scanner (Santa Clara, CA). The scanned results were compared with a wild-type p53 sequence. With the aid of a detection algorithm, mutations in the test samples were detected and assigned a scoring number based on the differences in hybridization intensity of every mutation. DNA from cell lines with known p53 gene mutation status was used as a control with each run. Twenty percent of the samples were repeated twice as a quality-control measure. Only mutations with a hybridization followed by scanning score of at least 13 were considered to be valid; a score of less than 13 indicates no evidence of mutation (14). To eliminate any false-positive results, confirmatory standard automated sequencing was performed on each mutation. Del = deletion; Ins = insertion.

 
Increased NOS-2 expression has been associated with several malignancies (19–22) and preneoplastic chronic inflammatory conditions (20,23–26). The overexpression of NOS-2 and NO in the non-neoplastic hepatic regenerative nodules—that are both preneoplastic and associated with increased risk of HCC—as presented here indicates a possible role in hepatic carcinogenesis. Although the frequency of NOS-2 overexpression in the non-neoplastic hemochromatosis tissues adjacent to the tumors is higher in our study than reported previously (57% versus 28%), the disease grade is different, because all of our samples were cirrhotic compared with 33% in our previous work (11). NO generated by NOS-2 has been reported to induce mutations in vitro and in animal models (27,28), and DNA damage by several mechanisms, such as nitrosamide deamination or induction of lipid peroxidation (28,29), as well as undermine cellular DNA repair processes performed by DNA glycosylase (30). In addition, ionic iron has been shown to modulate NO-mediated cell death and apoptosis (31). We have reported higher NOS-2 enzymatic activity in colonic adenomas than in colonic carcinomas (19). Furthermore, high levels of NOS-2 are associated with p53 mutations in colorectal neoplasms, particularly those with G : C to A : T transitions at CpG dinucleotides by a mechanism of NO-increased deamination of 5-methylcytosine (19,32). However, the p53 mutation spectrum in HCC associated with hemochromatosis reported here and elsewhere (15) is more consistent with some other unknown endogenous mutagenic mechanism that is mediated by lipid peroxidation, based on the types of mutations detected. The increase in etheno –dG and –dA DNA adducts, caused by lipid peroxidation in liver tissues from hemochromatosis patients (33,34), and the p53 mutational load (11) in liver tissue from individuals with hemochromatosis are both consistent with the hypothesis of generation of oxyradicals as an underlying mechanism of cancer in heavy metal overload diseases (16).

We thank Dorothea Dudek for editorial assistance.

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Manuscript received December 26, 2000; revised August 24, 2001; accepted September 4, 2001.


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