诱导型一氧化氮合酶，硝基酷氨酸在胃癌组织中表达与细胞凋亡的关系
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摘要
前言
胃癌是在我国发病率和死亡率均占第一位的恶性肿瘤。最近的一些研究发现，一氧化氮(Nitric Oxide，NO)在胃癌的发生和发展中起到重要作用。
NO是在NO合成酶(Nitric Oxide，NOS)催化下，由L-精氨酸氧化后生成的。NO在体内经氧化作用生成硝酸盐和亚硝酸盐。在特定的条件下，NO可以形成一种强力氧化物-过氧化亚硝酸盐(Peroxynitrite，ONOO~(2-))，其降解后产生一种活性氢氧根自由基(HOONO~-)，对细胞有毒性作用。过氧化亚硝酸盐和其降解产物将导致脂质的过氧化，以及导致调节酶功能及信号传导的酪氨酸分子的硝酸化，所有这些都将导致细胞的损伤。
NOS在组织中存在的形式是一族相关的但结构又有差异的同分异构体，即内皮型一氧化氮合酶(endothelial Nitric Oxide Synthase，eNOS)，神经型一氧化氮合酶(neural Nitric Oxide Synthase，nNOS)，还有诱导性一氧化氮合酶(inducible Nitric Oxide Synthase，iNOS)。eNOS和nNOS催化产生的NO水平较低，参与人体正常的生理反应，而iNOS则广泛存在于体内很多类型的细胞中，在病理条件下，一经诱导将产生大量的NO。大量的NO不但导致组织损伤、引发细胞的毒性作用，而且对细胞的生长和细胞的凋亡具有重要的调节作用。iNOS的诱导剂包括内毒素、γ-干扰素、白介素-1、TNF-α等。有研究证明，幽门螺旋杆菌(Helicobacter pylori)能够诱导炎症因子，如：IL-1，IL-6，IL-8，TNF-α的产生，而这些因子又能够影响iNOS的表达，人胃感染H．pylori后，胃癌的发生率增加了4-9倍，有超过60％的胃癌患者感染H．pylori。
以前的许多研究发现iNOS在肿瘤生长，浸润，和转移方面起到重要作用。许多肿瘤都表达iNOS。iNOS来源的NO即能抑制又能促进肿瘤的发展。这些相互矛盾的双重作用共同影响了肿瘤的临床病理和生物学特性。然而，在胃癌的发展过程中，iNOS起到的是抑制还是促进作用还没有明确报道。为此，本实验采用免疫组化的方法检测iNOS在胃癌组织中的表达和NO氧化反应的终产物之一-硝基酪氨酸(NT)在胃癌组织中的表达，并
通过检测胃癌组织中细胞凋亡与iNOS和NT的表达的关系来探讨训os的
表达与胃癌临床病理特点的关系，同时检查了H.刀肠ri感染对iNos的影
响，为阐述NO在胃癌发展中的作用提供实验依据。
方法
实验组66例胃癌标本和对照组10例正常胃组织均来自中国医科大学
附属第一医院肿瘤科胃癌手术标本，男性36例，女性30例，平均年龄57岁
(年龄范围29一78岁)，所有的研究标本都经过了大体和镜下检查，分化不
良型腺癌36例，分化型腺癌30例。所有研究对象在手术前均未接受化疗
和放疗。，rNM分期按照1997年UICC标准。用改良的McM通en’s染色对
胃组织的H.p郊ori感染进行检查。用免疫组化的方法探查iNos，NT在胃
癌组织和正常胃组织中的表达，为了检测胃癌组织中癌细胞的凋亡情况，实
验组和对照组病例均行脱氧核搪核昔酸末端转移酶介导的缺口末端标记
(钧NEL)染色。
结果
改良McM以len’s染色结果发现，H. pylori感染在胃癌组织中的发生
率为68 .18%，H.州口ri感染与iNOS的表达明显相关。免疫组织化学染色
显示，胃癌细胞中表达iNOS占62%，iNOS的表达水平与胃癌病期呈正相
关，其中，直径大于scm、TNMlll，W期的肿瘤、浸润深度为竹/T4及淋巴结
转移阳性的肿瘤，iNOS表达多呈强阳性，而与年龄，性别，组织分型，
uuren’s分型及Bo~a皿分型差异不显著。McNemar统计学分析证实，
iNOS和NT的表达呈正相关(P二0.25)。，rU NEL检测结果证明，66例标本
中，癌细胞凋亡的发生率为O一8.9%，均值为2.69%土2.61%，凋亡在中
高水平NT表达的肿瘤中的凋亡指数为4.09%土2.57%，明显高于阴性或
低表达NT的肿瘤(0.79%士0.95%)，M~一场飞itneyU检验亦证实，两者
之间差异显著(P<0 .001)。Sp即田man等级相关分析显示凋亡指数和NT表
达评分之间呈明显正相关(r二0.718，P<0.001)。
讨论
在过去的二十年里，研究发现，NO在肿瘤的发生和发展方面具有重要
的作用，对于NO的研究有可能揭开胃癌研究的新领域。本研究检查了iN-
05和NO氧化反应的终产物之一一硝基酪氨酸在胃癌中的表达，及其与胃
癌细胞凋亡之间的关系。
本实验结果表明，武05随着TNM分期的进展，淋巴结转移，肿瘤大小
和浸润深度而表达增加，既病期越晚，表达率越高，汹Os对胃癌的侵袭和进
展起到上调作用。肿瘤来源的班Q在肿瘤细胞的增殖，生存，转移和侵袭方
面表现了一种NO介导的多重作用。NO能够通过促进肿瘤细胞的转移，侵
袭和血管生成能力来刺激肿瘤的生长和转移。本实验还证明了H.刀lori
感染与胃癌组织里训05表达明显相关，提示H.尸了10“感染影响iNOS在胃
癌中的表达。
据报道，训05在很多肿瘤中均有表达。Jenkins等首先报道，人恶性肿
瘤细胞的水OScDNA片段转染鼠体细胞后出现了肿瘤的迅速增长现象。上
述实验也支持A血bS等的研究结果，在异种移植肿瘤细胞里，NO介导的
VEGF的上调与增加的血管密度一致。血管的增加不仅能促进肿瘤生长，
而且能提高肿瘤侵袭和转移的能力。距原发肿瘤?
Gastric carcinoma is one of the most common malignant diseases in the world, leading to the first cause of gastrointestinal cancer - related mortality. Recent studies showed that inducible nitric oxide synthase (iNOS) might play an important role in the growth of gastric cancer.
NO is derived from the oxidation of L - arginine catalyzed by Nitric Oxide Synthase ( NOS). In tissues NOS exisis mainly as a family of related but distinct isoforms, including endothelial ( eNOS) , neural ( nNOS) and inducible ( iNOS) isoforms. NO produced by eNOS and nNOS under normal physiology con-dictions is present at relatively low levels. In contrast, iNOS is widely distributed in a variety of cell types and upon induction can produce a high output of NO. Thus, excessive NO generation is important not only for its local destructive effect to tissues and cytotoxic to host cells, but also for the regulation of cell growth and programmed cell death ( apotosis). Epidemiological studies show that gastric cancer is closely linked to Helicobacter pylori ( Hp ) infection, because the incidence of gastric cancer increases 4-9 times after H. pylori infection , and more than 60% patients with gastric cancer had been infected with H. pylori , and H. pylori infection possesses a high risk for causing gastric canc
er.
Previous studies indicate that effects of NO synthesized by iNOS can be either tumor suppressing or tumor promoting. The tumor suppressing role has been identified by the typical findings in NO - mediated apoptosis, while the tumor promoting role has been demonstrated by the evidence that NO produced by iNOS may promote tumor angiogenesis and blood flow in tumor neovaculature, and enhance tumor growth, invasion, and metastasis. The conflicting dual roles of iNOS might work together to influence the clinical pathology and pathophysiology
of tumor.
However, when there is iNOS expression in gastric adenocarcinoma, the direction and condition in which the tumor may develop ( promoted or suppressed) , remain to be defined. Our study was designed to determine whether: (1) gastric adenocarcinoma cells express iNOS and NT; (2) the distribution of NT in gastric adenocarcinoma is related to the apoptosis; (3 ) presence of iNOS , NT and apoptosis correlate with clinical features of gastric adenocarcinoma. (4) H. pylori infection is associcated with the expression of iNOS.
Methods
Sixty - six Specimens of gastric adenocarcinoma and ten corresponding adjacent normal gastric tissues were obtained from surgically treated patients with gastric adenocarcinoma. Of them, 36 were male and 30 were female. The mean age was 57 years ( range, 29-78 years). All the selected adjacent control gastric tissues were identified as normal by gross and microscope study. As to the grade of tumor, 36 cases were classified as poorly differentiated adenocarcinoma, with 30 cases well and moderately differentiated. None of the 66 patients had received preoperative radiotherapy or chemotherapy, and all underwent curative surgical resection of the tumor along with regional lymph node dissection at least five years ago. Clinicopathologic characteristics of these patients were investigated based on the TNM classification of malignant tumors. A modified Mc-Mullen' s staining was used for the histological assessment of H. pylori infection. Immunohistochemistry was employed to localize iNOS and NT protein in normal and tumor tissues. The occurrence of apoptotic cell death ( apoptotic index [ AI ] ) was analyzed by the terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate biotin nick - end labeling ( TUNEL) method.
Result
Results showed that H. pylori infection was observed in 68. 18% of these specimens, the rate of H. pylori infection was there were significant positive cor-
relations between iNOS expression and H. pylori infection. iNOS expression was detected at an intermediate or high level in 41 of 66 (62% ) specimens of gastric adenocarcinoma; similarly, NT expression was 58% ; neither of them was found in the normal gastri
胃癌是在我国发病率和死亡率均占第一位的恶性肿瘤。最近的一些研究发现，一氧化氮(Nitric Oxide，NO)在胃癌的发生和发展中起到重要作用。
NO是在NO合成酶(Nitric Oxide，NOS)催化下，由L-精氨酸氧化后生成的。NO在体内经氧化作用生成硝酸盐和亚硝酸盐。在特定的条件下，NO可以形成一种强力氧化物-过氧化亚硝酸盐(Peroxynitrite，ONOO~(2-))，其降解后产生一种活性氢氧根自由基(HOONO~-)，对细胞有毒性作用。过氧化亚硝酸盐和其降解产物将导致脂质的过氧化，以及导致调节酶功能及信号传导的酪氨酸分子的硝酸化，所有这些都将导致细胞的损伤。
NOS在组织中存在的形式是一族相关的但结构又有差异的同分异构体，即内皮型一氧化氮合酶(endothelial Nitric Oxide Synthase，eNOS)，神经型一氧化氮合酶(neural Nitric Oxide Synthase，nNOS)，还有诱导性一氧化氮合酶(inducible Nitric Oxide Synthase，iNOS)。eNOS和nNOS催化产生的NO水平较低，参与人体正常的生理反应，而iNOS则广泛存在于体内很多类型的细胞中，在病理条件下，一经诱导将产生大量的NO。大量的NO不但导致组织损伤、引发细胞的毒性作用，而且对细胞的生长和细胞的凋亡具有重要的调节作用。iNOS的诱导剂包括内毒素、γ-干扰素、白介素-1、TNF-α等。有研究证明，幽门螺旋杆菌(Helicobacter pylori)能够诱导炎症因子，如：IL-1，IL-6，IL-8，TNF-α的产生，而这些因子又能够影响iNOS的表达，人胃感染H．pylori后，胃癌的发生率增加了4-9倍，有超过60％的胃癌患者感染H．pylori。
以前的许多研究发现iNOS在肿瘤生长，浸润，和转移方面起到重要作用。许多肿瘤都表达iNOS。iNOS来源的NO即能抑制又能促进肿瘤的发展。这些相互矛盾的双重作用共同影响了肿瘤的临床病理和生物学特性。然而，在胃癌的发展过程中，iNOS起到的是抑制还是促进作用还没有明确报道。为此，本实验采用免疫组化的方法检测iNOS在胃癌组织中的表达和NO氧化反应的终产物之一-硝基酪氨酸(NT)在胃癌组织中的表达，并
通过检测胃癌组织中细胞凋亡与iNOS和NT的表达的关系来探讨训os的
表达与胃癌临床病理特点的关系，同时检查了H.刀肠ri感染对iNos的影
响，为阐述NO在胃癌发展中的作用提供实验依据。
方法
实验组66例胃癌标本和对照组10例正常胃组织均来自中国医科大学
附属第一医院肿瘤科胃癌手术标本，男性36例，女性30例，平均年龄57岁
(年龄范围29一78岁)，所有的研究标本都经过了大体和镜下检查，分化不
良型腺癌36例，分化型腺癌30例。所有研究对象在手术前均未接受化疗
和放疗。，rNM分期按照1997年UICC标准。用改良的McM通en’s染色对
胃组织的H.p郊ori感染进行检查。用免疫组化的方法探查iNos，NT在胃
癌组织和正常胃组织中的表达，为了检测胃癌组织中癌细胞的凋亡情况，实
验组和对照组病例均行脱氧核搪核昔酸末端转移酶介导的缺口末端标记
(钧NEL)染色。
结果
改良McM以len’s染色结果发现，H. pylori感染在胃癌组织中的发生
率为68 .18%，H.州口ri感染与iNOS的表达明显相关。免疫组织化学染色
显示，胃癌细胞中表达iNOS占62%，iNOS的表达水平与胃癌病期呈正相
关，其中，直径大于scm、TNMlll，W期的肿瘤、浸润深度为竹/T4及淋巴结
转移阳性的肿瘤，iNOS表达多呈强阳性，而与年龄，性别，组织分型，
uuren’s分型及Bo~a皿分型差异不显著。McNemar统计学分析证实，
iNOS和NT的表达呈正相关(P二0.25)。，rU NEL检测结果证明，66例标本
中，癌细胞凋亡的发生率为O一8.9%，均值为2.69%土2.61%，凋亡在中
高水平NT表达的肿瘤中的凋亡指数为4.09%土2.57%，明显高于阴性或
低表达NT的肿瘤(0.79%士0.95%)，M~一场飞itneyU检验亦证实，两者
之间差异显著(P<0 .001)。Sp即田man等级相关分析显示凋亡指数和NT表
达评分之间呈明显正相关(r二0.718，P<0.001)。
讨论
在过去的二十年里，研究发现，NO在肿瘤的发生和发展方面具有重要
的作用，对于NO的研究有可能揭开胃癌研究的新领域。本研究检查了iN-
05和NO氧化反应的终产物之一一硝基酪氨酸在胃癌中的表达，及其与胃
癌细胞凋亡之间的关系。
本实验结果表明，武05随着TNM分期的进展，淋巴结转移，肿瘤大小
和浸润深度而表达增加，既病期越晚，表达率越高，汹Os对胃癌的侵袭和进
展起到上调作用。肿瘤来源的班Q在肿瘤细胞的增殖，生存，转移和侵袭方
面表现了一种NO介导的多重作用。NO能够通过促进肿瘤细胞的转移，侵
袭和血管生成能力来刺激肿瘤的生长和转移。本实验还证明了H.刀lori
感染与胃癌组织里训05表达明显相关，提示H.尸了10“感染影响iNOS在胃
癌中的表达。
据报道，训05在很多肿瘤中均有表达。Jenkins等首先报道，人恶性肿
瘤细胞的水OScDNA片段转染鼠体细胞后出现了肿瘤的迅速增长现象。上
述实验也支持A血bS等的研究结果，在异种移植肿瘤细胞里，NO介导的
VEGF的上调与增加的血管密度一致。血管的增加不仅能促进肿瘤生长，
而且能提高肿瘤侵袭和转移的能力。距原发肿瘤?
Gastric carcinoma is one of the most common malignant diseases in the world, leading to the first cause of gastrointestinal cancer - related mortality. Recent studies showed that inducible nitric oxide synthase (iNOS) might play an important role in the growth of gastric cancer.
NO is derived from the oxidation of L - arginine catalyzed by Nitric Oxide Synthase ( NOS). In tissues NOS exisis mainly as a family of related but distinct isoforms, including endothelial ( eNOS) , neural ( nNOS) and inducible ( iNOS) isoforms. NO produced by eNOS and nNOS under normal physiology con-dictions is present at relatively low levels. In contrast, iNOS is widely distributed in a variety of cell types and upon induction can produce a high output of NO. Thus, excessive NO generation is important not only for its local destructive effect to tissues and cytotoxic to host cells, but also for the regulation of cell growth and programmed cell death ( apotosis). Epidemiological studies show that gastric cancer is closely linked to Helicobacter pylori ( Hp ) infection, because the incidence of gastric cancer increases 4-9 times after H. pylori infection , and more than 60% patients with gastric cancer had been infected with H. pylori , and H. pylori infection possesses a high risk for causing gastric canc
er.
Previous studies indicate that effects of NO synthesized by iNOS can be either tumor suppressing or tumor promoting. The tumor suppressing role has been identified by the typical findings in NO - mediated apoptosis, while the tumor promoting role has been demonstrated by the evidence that NO produced by iNOS may promote tumor angiogenesis and blood flow in tumor neovaculature, and enhance tumor growth, invasion, and metastasis. The conflicting dual roles of iNOS might work together to influence the clinical pathology and pathophysiology
of tumor.
However, when there is iNOS expression in gastric adenocarcinoma, the direction and condition in which the tumor may develop ( promoted or suppressed) , remain to be defined. Our study was designed to determine whether: (1) gastric adenocarcinoma cells express iNOS and NT; (2) the distribution of NT in gastric adenocarcinoma is related to the apoptosis; (3 ) presence of iNOS , NT and apoptosis correlate with clinical features of gastric adenocarcinoma. (4) H. pylori infection is associcated with the expression of iNOS.
Methods
Sixty - six Specimens of gastric adenocarcinoma and ten corresponding adjacent normal gastric tissues were obtained from surgically treated patients with gastric adenocarcinoma. Of them, 36 were male and 30 were female. The mean age was 57 years ( range, 29-78 years). All the selected adjacent control gastric tissues were identified as normal by gross and microscope study. As to the grade of tumor, 36 cases were classified as poorly differentiated adenocarcinoma, with 30 cases well and moderately differentiated. None of the 66 patients had received preoperative radiotherapy or chemotherapy, and all underwent curative surgical resection of the tumor along with regional lymph node dissection at least five years ago. Clinicopathologic characteristics of these patients were investigated based on the TNM classification of malignant tumors. A modified Mc-Mullen' s staining was used for the histological assessment of H. pylori infection. Immunohistochemistry was employed to localize iNOS and NT protein in normal and tumor tissues. The occurrence of apoptotic cell death ( apoptotic index [ AI ] ) was analyzed by the terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate biotin nick - end labeling ( TUNEL) method.
Result
Results showed that H. pylori infection was observed in 68. 18% of these specimens, the rate of H. pylori infection was there were significant positive cor-
relations between iNOS expression and H. pylori infection. iNOS expression was detected at an intermediate or high level in 41 of 66 (62% ) specimens of gastric adenocarcinoma; similarly, NT expression was 58% ; neither of them was found in the normal gastri
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20. Morris SM jr, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol 1994; 266:E829-839.
21. Blaser M J, Perez -Perez GI; Kleanthous H, Cover TL, Peek RM, Chyou PH, Stemmermann GN, Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 1995; 55: 2111 - 2115.
22. Dong Z, Staroselsky AH, Qi X, Xie K, Fidler IJ. Inverse correlation between expression of inducible nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells. Cancer Res 1994 ;54:789-793.
23. Albina JE, Abate JA, Henry WL Jr. Nitric oxide production is required for routine resident peritoneal macrophages to suppress mitogen -stimulated T cell proliferation. Role of IFN - gamma in the induction of the nitric oxide -synthesizing pathway. J Immunol 1991; 147:144-148.
24. Quan J, Fan XG. The progress of experiment for Helicobacter pylori infection and gastric carcinogenesis. Shijie Huaren Xiaohua Zazhi 1999; 7:1068 - 1069.
25. Vickers SM, MacMillan - Crow LA, Green M, Ellis C, Thompson JA. Association of increased immunostaining for inducible nitric oxide synthase and nitrotyrosine with fibroblast growth factor transformation in pancreatic cancer. Arch Surg 1999;134:245-251.
26. Sonoki T, Matsuzaki H, Nagasaki A, Hata H, Yoshida M, Matsuoka M, Kuribayashi N, Kimura T, Harada N, Takatsuki K, Mitsuya H, Mori M. Detection of inducible nitric oxide synthase (iNOS) mRNA by RT - PCR in ATL patients and HTLV-Ⅰ infected cell lines: clinical features and apoptosis by NOS inhibitor. Leukemia 1999;13:713 -718.
27. Kojima M, Morisaki T, Tsukahara Y, Uchiyama A, Matsunari Y, Mibu R, Tanaka M. Nitric oxide synthase expression and nitric oxide production in human colon carcinoma tissue. J Surg Oncol 1999; 70:222 -229.
28. Ambs S, Hussain SP, Harris CC. Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. ASEB J 1997; 11: 443 - 448.
29. Dimmeler S, Zeiher AM. Nitric oxide and apoptosis: another paradigm for the double - edged role of nitric oxide. Nitric Oxide 1997; 1: 275 -281.
30. Brune B, yon Knethen A, Sandau KB. Nitric oxide and its role in apoptosis. Eur J Pharmacol 1998; 351: 261 - 272.
31. Sobin LH, Fleming ID. TNM Classification of Malignant Tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997; 80:1803 -1804.
32. Iwaki H, Sugiyama T, Asaka M. A modified McMullen's stainingfor Helicobacter pylori: a high - contrast, visibly prominent method. Helicobacter 1998; 3:45-48.
33. Krajewska M, Krajewski S, Epstein JI, Shabaik A, Sauvageot J, Song K, Kitada S, Reed JC. Immunohistochemical analysis of bc1-2, bax, bcl-X, and mcl - 1 expression in prostate cancers. Am J Pathol 1996;148: 1567-1576.
34. Ansari B, Coates PJ, Greenstein BD, Hall PA. In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 1993; 170: 1-8.
35. Koh E, Noh SH, Lee YD, Lee HY, Han JW, Lee HW, Hong S. Differential expression of nitric oxide synthase in human stomach cancer. Cancer Lett 1999; 146: 173 - 180.
36. Doi C, Noguchi Y, Marat D, Saito A, Fukuzawa K, Yoshikawa T, Tsuburaya A, Ito T. Expression of nitric oxide synthase in gastric cancer. Cancer Lett 1999; 144: 161 - 167.
37. Mannick EE, Bravo I.F., Zarama G, Realpe JL, Zhang XJ, Ruiz B, Fontham ET, Mera R, Miller M J, Correa P.. Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Hehcobacter pylori gastritis: effect of antibiotics and antioxidants. Cancer Res. 1996 Jul 15; 56(14): 3238-43.
38. Goto T, Haruma K, Kitadai Y, Ito M, Yoshihara M, Sumii K, Hayakawa N, Kajiyama G. Enhanced expression of inducible nitric oxide synthase and nitrotyrosine in gastric mucosa of gastric cancer patients. Clin Cancer Res. 1999 Jun;5(6): 1411-5.
39. Yagihashi N, Kasajima H, Sugai S, Matsumoto K, Ebina Y, Morita T, Murakami T, Yagihashi S. Increased in situ expression of nitric oxide synthase in human colorectal cancer. Virchows Arch 2000; 436: 109 - 114.
40. Wolf H, Haeckel C, Roessner A. Inducible nitric oxide synthase expression in human urinary bladder cancer. Virchows Arch 2000; 437:662-666.
41. Thomsen LL, Lawton FG, Knowles RG, et al. Nitric oxide synthase activity in human gynecological cancer. Cancer Res, 1994, 54:1352-1354.
42. Vakkala M, Kahlos K, Lakari E, Paakko P, Kinnula V, Soini Y. Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas. Clin Cancer Res 2000; 6:2408-2416.
43. Thomsen LL, Miles DW, Happerfield L, et al. Nitric oxide synthase activi-
ty in human breast cancer. Br J Cancer, 1995, 72:41-44.
44. Duenas - Gonzalez A, Isales CM, del Mar Abad - Hernandez M, et al. Expression of inducible nitric oxide synthase in breast cancer correlates with metastatic disease. Mod Pathol, 1997, 10: 645 - 649.
45. Cobbs CS, Brenman JE, Aldape KD, et al. Expression of nitric oxide synthase in human centralnervous system tumors. Cancer Res 1995; 55:727-730.
46. Jenkins DC, Charles IG, Thomsen LL, et al. Roles of nitric oxide in tumour growth. Proc Nail Acad Sci USA, 1995, 92:4392-4396.
47. Ambs S, Merriam WG, Ogunfusika MO, et al. p53 and vascular endothelial growth factor regulate tumor growth of NOS2 - expressing human carcinoma cells. Nat Med, 1998, 4: 1371 - 1376.
48. Rocha M, Kruger A, Van Rooijen N, Schirrmacher V, Umansky V. Liver endothelial cells participate in T -cell -dependent host resistance to lymphoma metastasis by production of nitric oxide in vivo. Int J Cancer, 1995, 63: 405 -411.
49. Nguyen T, Brunson D, Crespi CL, Penman BW, Wishnok JS, Tannenbaum SR. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc Nail Acad Sci U S A. 1992; 89:3030-3034.
50. Tamir S, deRojas - Walker T, Wishnok JS, Tannenbaum SR. DNA damage and genotoxicity by nitric oxide. Methods Enzymol 1996; 269:230-243.
51. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 1996;313: 17-29.
52. Graziewicz M, Wink DA, Laval F. Nitric oxide inhibits DNA ligase activity: potential mechanisms for NO-mediated DNA damage. Carcinogenesis 1996; 17: 2501 - 2505.
53. Lejeune P, Lagadec P, Onier N, Pinard D, Ohshima H, Jeannin JF Nitric oxide involvement in tumor- induced immunosuppression. J Immunol 1994; 152: 5077 - 5083.
54. Takahashi Y, Bucana CD, Akagi Y, Liu W, Cleary KR, Mai M, Ellis LM. Significance of platelet -derived endothelial cell growth factor in the
angiogenesis of human gastric cancer. Clin Cancer Res 1998; 4:429-434.
55. Dachs GU, Tozer GM. Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer, 2000, 36:1649-1660.
56. Lala PK. Significance of nitric oxide in carcinogenesis, tumor progression and cancer therapy. Cancer Metastasis Rev 1998; 17: 1-6.
57. Juang SH, Xie K, Xu L, Shi Q, Wang Y, Yoneda J, Fidler IJ. Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the routine inducible nitric oxide synthase gene. Hum Gene Ther 1998; 9: 845-854.
58. Drapier JC, Hibbs JB Jr. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine - dependent inhibition of mitochondrial iron -sulfur enzymes in the macrophage effector cells. J Immunol, 1988, 140: 2829-2838.
59. Doyle, MP, Hoekstra,JW. Oxidation of nitrogen oxides by bound dioxygen in hemoproteins. J Inorg Biochem, 1981, 14:351-358.
60. Kim YM, Bombeck CA, Billiar TR. Nitric oxide as a bifunctional regulator of apoptosis Circ Res 1999; 84:253-256.
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16. Geller DA, Nussler AK, Di Silvio M, Lowenstein C J, Shapiro RA, Wang SC, Simmons RL, Billiar TR. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A 1993 ;90:522-526.
17. Kondo S, Toyokuni S, Iwasa Y, Tanaka T, Onodera H, Hiai H, Imamura M. Persistent oxidative stress in human colorectal carcinoma, but not in adenoma. Free Radic Biol Med 1999;27:401 -410.
18. Gavrieli Y, Sherman Y, Ben - Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493 - 501.
19. Chartrain NA, Geller DA, Koty PP,Sitrin NF, Nussler AK, Hoffman EP, Billiar TR, Hutchinson NI, Mudgett JS. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J Biol Chem 1994; 269:6765-6772.
20. Morris SM jr, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol 1994; 266:E829-839.
21. Blaser M J, Perez -Perez GI; Kleanthous H, Cover TL, Peek RM, Chyou PH, Stemmermann GN, Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 1995; 55: 2111 - 2115.
22. Dong Z, Staroselsky AH, Qi X, Xie K, Fidler IJ. Inverse correlation between expression of inducible nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells. Cancer Res 1994 ;54:789-793.
23. Albina JE, Abate JA, Henry WL Jr. Nitric oxide production is required for routine resident peritoneal macrophages to suppress mitogen -stimulated T cell proliferation. Role of IFN - gamma in the induction of the nitric oxide -synthesizing pathway. J Immunol 1991; 147:144-148.
24. Quan J, Fan XG. The progress of experiment for Helicobacter pylori infection and gastric carcinogenesis. Shijie Huaren Xiaohua Zazhi 1999; 7:1068 - 1069.
25. Vickers SM, MacMillan - Crow LA, Green M, Ellis C, Thompson JA. Association of increased immunostaining for inducible nitric oxide synthase and nitrotyrosine with fibroblast growth factor transformation in pancreatic cancer. Arch Surg 1999;134:245-251.
26. Sonoki T, Matsuzaki H, Nagasaki A, Hata H, Yoshida M, Matsuoka M, Kuribayashi N, Kimura T, Harada N, Takatsuki K, Mitsuya H, Mori M. Detection of inducible nitric oxide synthase (iNOS) mRNA by RT - PCR in ATL patients and HTLV-Ⅰ infected cell lines: clinical features and apoptosis by NOS inhibitor. Leukemia 1999;13:713 -718.
27. Kojima M, Morisaki T, Tsukahara Y, Uchiyama A, Matsunari Y, Mibu R, Tanaka M. Nitric oxide synthase expression and nitric oxide production in human colon carcinoma tissue. J Surg Oncol 1999; 70:222 -229.
28. Ambs S, Hussain SP, Harris CC. Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. ASEB J 1997; 11: 443 - 448.
29. Dimmeler S, Zeiher AM. Nitric oxide and apoptosis: another paradigm for the double - edged role of nitric oxide. Nitric Oxide 1997; 1: 275 -281.
30. Brune B, yon Knethen A, Sandau KB. Nitric oxide and its role in apoptosis. Eur J Pharmacol 1998; 351: 261 - 272.
31. Sobin LH, Fleming ID. TNM Classification of Malignant Tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997; 80:1803 -1804.
32. Iwaki H, Sugiyama T, Asaka M. A modified McMullen's stainingfor Helicobacter pylori: a high - contrast, visibly prominent method. Helicobacter 1998; 3:45-48.
33. Krajewska M, Krajewski S, Epstein JI, Shabaik A, Sauvageot J, Song K, Kitada S, Reed JC. Immunohistochemical analysis of bc1-2, bax, bcl-X, and mcl - 1 expression in prostate cancers. Am J Pathol 1996;148: 1567-1576.
34. Ansari B, Coates PJ, Greenstein BD, Hall PA. In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 1993; 170: 1-8.
35. Koh E, Noh SH, Lee YD, Lee HY, Han JW, Lee HW, Hong S. Differential expression of nitric oxide synthase in human stomach cancer. Cancer Lett 1999; 146: 173 - 180.
36. Doi C, Noguchi Y, Marat D, Saito A, Fukuzawa K, Yoshikawa T, Tsuburaya A, Ito T. Expression of nitric oxide synthase in gastric cancer. Cancer Lett 1999; 144: 161 - 167.
37. Mannick EE, Bravo I.F., Zarama G, Realpe JL, Zhang XJ, Ruiz B, Fontham ET, Mera R, Miller M J, Correa P.. Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Hehcobacter pylori gastritis: effect of antibiotics and antioxidants. Cancer Res. 1996 Jul 15; 56(14): 3238-43.
38. Goto T, Haruma K, Kitadai Y, Ito M, Yoshihara M, Sumii K, Hayakawa N, Kajiyama G. Enhanced expression of inducible nitric oxide synthase and nitrotyrosine in gastric mucosa of gastric cancer patients. Clin Cancer Res. 1999 Jun;5(6): 1411-5.
39. Yagihashi N, Kasajima H, Sugai S, Matsumoto K, Ebina Y, Morita T, Murakami T, Yagihashi S. Increased in situ expression of nitric oxide synthase in human colorectal cancer. Virchows Arch 2000; 436: 109 - 114.
40. Wolf H, Haeckel C, Roessner A. Inducible nitric oxide synthase expression in human urinary bladder cancer. Virchows Arch 2000; 437:662-666.
41. Thomsen LL, Lawton FG, Knowles RG, et al. Nitric oxide synthase activity in human gynecological cancer. Cancer Res, 1994, 54:1352-1354.
42. Vakkala M, Kahlos K, Lakari E, Paakko P, Kinnula V, Soini Y. Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas. Clin Cancer Res 2000; 6:2408-2416.
43. Thomsen LL, Miles DW, Happerfield L, et al. Nitric oxide synthase activi-
ty in human breast cancer. Br J Cancer, 1995, 72:41-44.
44. Duenas - Gonzalez A, Isales CM, del Mar Abad - Hernandez M, et al. Expression of inducible nitric oxide synthase in breast cancer correlates with metastatic disease. Mod Pathol, 1997, 10: 645 - 649.
45. Cobbs CS, Brenman JE, Aldape KD, et al. Expression of nitric oxide synthase in human centralnervous system tumors. Cancer Res 1995; 55:727-730.
46. Jenkins DC, Charles IG, Thomsen LL, et al. Roles of nitric oxide in tumour growth. Proc Nail Acad Sci USA, 1995, 92:4392-4396.
47. Ambs S, Merriam WG, Ogunfusika MO, et al. p53 and vascular endothelial growth factor regulate tumor growth of NOS2 - expressing human carcinoma cells. Nat Med, 1998, 4: 1371 - 1376.
48. Rocha M, Kruger A, Van Rooijen N, Schirrmacher V, Umansky V. Liver endothelial cells participate in T -cell -dependent host resistance to lymphoma metastasis by production of nitric oxide in vivo. Int J Cancer, 1995, 63: 405 -411.
49. Nguyen T, Brunson D, Crespi CL, Penman BW, Wishnok JS, Tannenbaum SR. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc Nail Acad Sci U S A. 1992; 89:3030-3034.
50. Tamir S, deRojas - Walker T, Wishnok JS, Tannenbaum SR. DNA damage and genotoxicity by nitric oxide. Methods Enzymol 1996; 269:230-243.
51. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 1996;313: 17-29.
52. Graziewicz M, Wink DA, Laval F. Nitric oxide inhibits DNA ligase activity: potential mechanisms for NO-mediated DNA damage. Carcinogenesis 1996; 17: 2501 - 2505.
53. Lejeune P, Lagadec P, Onier N, Pinard D, Ohshima H, Jeannin JF Nitric oxide involvement in tumor- induced immunosuppression. J Immunol 1994; 152: 5077 - 5083.
54. Takahashi Y, Bucana CD, Akagi Y, Liu W, Cleary KR, Mai M, Ellis LM. Significance of platelet -derived endothelial cell growth factor in the
angiogenesis of human gastric cancer. Clin Cancer Res 1998; 4:429-434.
55. Dachs GU, Tozer GM. Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer, 2000, 36:1649-1660.
56. Lala PK. Significance of nitric oxide in carcinogenesis, tumor progression and cancer therapy. Cancer Metastasis Rev 1998; 17: 1-6.
57. Juang SH, Xie K, Xu L, Shi Q, Wang Y, Yoneda J, Fidler IJ. Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the routine inducible nitric oxide synthase gene. Hum Gene Ther 1998; 9: 845-854.
58. Drapier JC, Hibbs JB Jr. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine - dependent inhibition of mitochondrial iron -sulfur enzymes in the macrophage effector cells. J Immunol, 1988, 140: 2829-2838.
59. Doyle, MP, Hoekstra,JW. Oxidation of nitrogen oxides by bound dioxygen in hemoproteins. J Inorg Biochem, 1981, 14:351-358.
60. Kim YM, Bombeck CA, Billiar TR. Nitric oxide as a bifunctional regulator of apoptosis Circ Res 1999; 84:253-256.