Association between inducible nitric oxide synthase expression and p53 status in human esophageal squamous cell carcinoma

Nitric oxide (NO) produced by the NO synthase (NOS), a family of enzymes such as inducible NOS (iNOS), has been suggested to play an important role in tumor bioloty. We immunohistochemically examined iNOS and p53 protein expression in 105 patients with esophageal squamous cell carcinoma (ESCC). Direct sequence analysis for the p53 gene was performed in 51 of 105 tumors. In total, 56 of 105 (53.3%) tumors exhibited intracytoplasmic staining for anti-iNOS antibody, including 17 (16.2%) cases of homogeneous and intense immunostaining (++) and 39 (37.1%) of heterogeneous staining (+). Of 62 p53 protein-positive tumors, 40 (63.5%) were positive for iNOS, and of 43 p53 protein-negative tumors, 27 (62.8%) were negative for iNOS. Of 34 iNOS-positive tumors, 23 (67.6%) carried a p53 gene mutation, and of 17 iNOS-negative tumors, 12 (70.6%) had wild-type p53 gene. There was a significant relationship between iNOS immunoreactivity and p53 protein overexpression (p = 0.0058) as well as p53 mutation frequency (p = 0.0163). No association was found between iNOS immunoreactivity and p53 mutation type, any clinicopathological factor and patient prognosis. Our in vivo findings suggest that iNOS activity might be associated with p53 alteration and contribute to tumorigenesis in ESCC.     

p53 and human papillomavirus dna in renal pelvic and ureteral carcinoma including dysplastic lesions

Ninety-eight cases of transitional-cell carcinoma (TCC) of the renal pelvis and ureter, including dysplastic lesions, were studied for tumor incorporation of human papillomavirus (HPV) type-16 and type-18 DNA by in situ hybridization (ISH) with DNA probes for each HPV viral type. Immunohistochemical analysis of p53 expression was also performed. Fresh tumor tissues from 26 patients were also studied for p53 mutations in exons 4 through 9 by direct sequencing and for HPV infection by polymerase chain reaction (PCR). Thirty-two tumors were positive for HPV DNAs, including 6 double-positive cases. Among these tumors, adjacent dysplastic lesions in 21 cases (66%) also revealed identical reactivity. Overexpressed p53 was detected in 26 cases. Expression of p53 was also detected in dysplastic lesions in 19 out of these 26 cases (73%). Three cases were positive for both HPV DNA and p53 antibody. p53 point mutation was detected in 7 of 26 cases, 6 of which were also positive for p53. HPV type-16 DNA was detected in 6 cases by PCR, 4 of which were also ISH-positive. Overexpressed p53 was frequently detected in invasive and non-papillary tumors (p < 0.01) and in high-grade tumors (p < 0.05). HPV infection was more common in non-invasive and papillary tumors (p < 0.05). These findings suggest that HPV infection or overex-pression (mutation) of p53 may be an early event and be related to phenotypes of tumor-cell growth patterns and progression. © 1995 Wiley-Liss, Inc.     

Expression of inducible nitric oxide synthase is significantly correlated with expression of vascular endothelial growth factor and dendritic cell infiltration in patients with advanced gastric carcinoma

OBJECTIVE: Nitric oxide (NO) is a product of L-arginine to L-citrulline conversion by nitric oxide synthase (NOS). The inducible form of NOS (iNOS) is one of three classes of NOS and the strongest producer of NO. It has been reported that NO correlates with angiogenesis and immune responses in some types of cancer, however, the correlations between iNOS expression, angiogenesis, and immune responses are still unclear in gastric carcinoma. METHODS: iNOS expression was determined in 135 gastric cancer patients by immunohistochemical procedures and compared with the expression of vascular endothelial growth factor (VEGF), microvessel (MV) density, and dendritic cell (DC) infiltration to evaluate the effect of iNOS on angiogenesis and immune responses in gastric carcinoma. RESULTS: iNOS expression was detected in 106 (78.5%) of 135 cases. There was a close correlation between iNOS expression and VEGF expression, a correlation with MV density and an inverse correlation with DC infiltration. There was no correlation between iNOS and p53 expression. The prognoses of patients whose tumors expressed iNOS were significantly worse than those of patients whose tumors did not express iNOS. Multivariate analysis indicated iNOS expression was an independent prognostic factor. CONCLUSION: iNOS might be associated with tumor progression by stimulating angiogenesis and suppressing immune responses in gastric carcinoma.     

Overexpressed cyclin D1 protein related to p53 and retinoblastoma proteins in transitional cell carcinoma of the urinary tract

We employed immunohistochemical methods to investigate the overexpression of cyclin D1 protein as well as p53 protein and retinoblastoma protein (pRb) in 57 patients with transitional cell carcinoma (TCC) of the urinary tract, in order to determine their frequency and significance, and also association with the clinicopathological behavior of this tumor. Nuclear immunostaining with cyclin D1 antibody revealed 7 homogeneously stained tumors (12.3%), including 4 heterogeneous pRb expressions, of which only one was also positive for p53. Positive immunostaining with each antibody to p53 or pRb was obtained in 15 (26.3%) and 49 (86%) tumors, respectively. The 49 pRb-positive tumors included 6 homogeneous and 43 heterogeneous nuclear immunostaining patterns. The remaining pRb-negative 8 tumors included 5 cyclin D1-negative tumors. The prevalence of p53 staining was higher in high-grade (P<0.05) and invasive types of tumors (P<0.05) than the other types studied. On the other hand, no significant relationship was found between cyclin D1- or pRb-immunoreactivity and clinicopathological factors. These findings provide evidence for infrequent cyclin D1 protein overexpression in TCC independently associated with p53 protein and/or pRb overexpression.     

Prognostic significance of cyclin E and p53 protein overexpression in carcinoma of the renal pelvis and ureter.

Cyclin E gene alteration in the cell cycle plays an important role in carcinogenesis, while p53 protein affects different phase checkpoint pathways by activating p21WAF1/CIP1 in the normal cell cycle. We immunohistochemically examined the expression of cyclin E and p53 proteins in 121 patients with transitional cell carcinoma (TCC) of the renal pelvis and ureter to determine their significance for tumour behaviour and patient prognosis. Cyclin E and p53 immunostaining of the nucleus was observed in 36 tumours (29.8%) and 35 tumours (28.9%) respectively. A significant percentage, 69.4% (25 out of 36 tumours), of the cyclin E-positive tumours exhibited simultaneous labelling for p53 (P < 0.05). Mirror-section technique was performed in five selected double-positive tumours to identify cancer cells that were nuclei positive for both cyclin E and p53. The prevalence of cases simultaneously exhibiting both cyclin E and p53 immunostaining was higher in the high-grade tumours (P < 0.01) than in the other types of tumours. Patients with TCCs coexpressing cyclin E and p53 had a significantly poorer prognosis than those expressing neither cyclin E nor p53 (P < 0.001). These in vivo findings provide evidence for cyclin E protein overexpression in TCCs intimately associated with p53 alteration and suggest that simultaneous overexpression of both cyclin E and p53 is related to tumour behaviour and poor prognosis.     

粘着斑激酶在膀胱移行细胞癌中表达的意义

目的 :探讨粘着斑激酶 (FAK)在膀胱移行细胞癌 (TCC)中的表达与肿瘤分化、浸润的关系 ,以及与p53、bcl 2、ki 67表达的关系。方法 :采用免疫组织化学方法 ,测定FAK在 81例膀胱TCC中的表达情况。结果 :FAK在膀胱癌组织中的表达明显高于癌旁黏膜 ,浸润性膀胱癌明显高于非浸润性 ,p53或ki 67阳性膀胱癌明显高于阴性组。结论 :FAK可能是一种肿瘤转化相关酶 ,又是一种肿瘤演变相关酶 ;它的表达量可作为膀胱癌发生发展过程中有价值的指标。     

基质金属蛋白酶与膀胱癌分化和浸润的关系

目的 探讨基质金属蛋白酶2（MMP－2）在膀胱移行细胞癌（TCC）中的表达与肿瘤分化和浸润,以及与p53和ki-67表达的关系。方法 采用免疫组化DAKO En Vision System方法,测定MMP－2,p53和ki-67在83例膀胱TCC中的表达。结果 MMP－2在TCC中的性率为63．9％,表达显著高于癌旁粘膜（P＜0．01）;MMP－2表达强度与TCC癌细胞的分化高低和浸润深度呈明显正相关（P＜0．05）,并与ki-67表达有显著意义的关联（P＜0．01）,但与p53表达无明显相关（P＞0．05）。结论 MMP－2在TCCR的发展和浸润过程中发挥重要作用,可能成为预测TCC预后的参考指标。     

p53 protein accumulation and genomic instability in head and neck multistep tumorigenesis.

Head and neck cancer develops in a multistep process and is associated with increasing frequencies of p53 alterations and with increasing genomic instability. To study the relationship of p53 alterations and genomic instability during head and neck tumorigenesis, we analyzed p53 protein expression and chromosome 9 and 17 polysomy in 48 squamous cell carcinomas of the head and neck and their adjacent normal epithelium (31 sites), hyperplastic (24 sites), and dysplastic lesions (26 sites). Normal oral epithelium obtained from seven nonsmoking, cancer-free individuals served as negative controls. Six (19%) of 31 lesions in adjacent normal epithelium, 7 (29%) of 24 hyperplastic lesions, 12 (46%) of 26 dysplastic lesions, and 28 (58%) of 48 squamous cell carcinomas expressed p53. In contrast, no normal control epithelium had detectable p53 expression. To determine the relationship between dysregulated p53 expression and genomic instability during tumorigenesis, we compared p53 immunohistochemistry distributions and chromosome polysomy levels (by chromosome in situ hybridization) in different histological groups associated with tissue progression. Although the degree of chromosome polysomy increased for all of the groups during histological progression, lesions with dysregulated p53 expression showed nearly 2-4-fold increased levels of chromosome polysomy. This trend was significant for dysplastic lesions (P = 0.005 and P = 0.002 for chromosomes 9 and 17, respectively) and for squamous cell carcinoma (P = 0.005 and P = 0.002 for chromosomes 9 and 17, respectively). Image analysis studies for 28 p53-expressing tumors and their adjacent premalignant lesions demonstrated a strong spatial correlation between stepwise transitions from low to high p53 expression and increased chromosome polysomy frequencies in 13 (46%) of 28 cases. These findings suggest that altered p53 expression is associated with increased genetic instability in preneoplastic epithelium and may play a driving force for increasing the rate of accumulation of genetic events during head and neck tumorigenesis.     

Different combinations of genetic/epigenetic alterations inactivate the p53 and pRb pathways in invasive human bladder cancers

Inactivation of both the pRb (pRb-cyclin D1/cyclin-dependent kinase 4/6-p16) and p53 (p53-p21(WAF1)-p14(ARF)) pathways is thought to be essential for immortalization in vitro and malignant transformation in vivo. We identified different combinations of pRb and p53 pathway alterations in 12 invasive transitional cell carcinomas (TCCs) and addressed the functional significance of the different combinations observed. Results showed four combinations of alterations including -pRb/-p53 (ie., pRb inactivated in the pRb pathway and p53 inactivated in the p53 pathway; four TCCs), -p16/-p53 (four TCCs), -p16/-p21(WAF1) (one TCC), and -p16/ -p14(ARF) (two TCCs). These groups include two new combinations (ie., -p16/-p53 and -p16/-p21(WAF1)) not reported previously for TCCs. An alteration in the key components of the p53 pathway was not detected in one invasive TCC that had inactivated p16. Note that all four TCCs with inactivated pRb had mutant p53; thus, the combinations of -pRb/ -p21(WAF1) and -pRb/-p14(ARF) were not observed. Only two of eight TCCs with altered p16 had concomitant p14(ARF) loss, demonstrating that simultaneous inactivation of these two 9p21INK4a tumor suppressor genes is not obligatory. To determine the biological phenotypes of TCCs with different combinations of pRb and p53 pathway alterations, their downstream responses to gamma radiation were studied in vitro. As expected, none of eight TCCs with mutant p53 responded to gamma radiation by elevation of p53, p21(WAF1), or mdm2 or by cell cycle arrest. Only two of four TCCs with wild-type p53 and wild-type pRb (the combination of -p16/-p14(ARF)) showed normal downstream responses to gamma radiation and underwent cell cycle arrest. Two TCCs with wild-type pRb and wild-type p53 (the combination of -pl6/-p21(WAF1) and one TCC with -p16) failed to show cell cycle arrest in response to radiation. This was attributed to the absence of p21(WAF1) in one TCC. In summary, these data support a model of invasive bladder cancer pathogenesis in which both the pRb and p53 pathways are usually inactivated and the biology of the tumor is impacted by the mechanism of their inactivations.     

Assessment of cell cycle-related elements p53, p21WAF1/Cip1, cyclin D1 and PCNA in a mixed transitional cell carcinoma and adenocarcinoma of the renal pelvis: a case report

A case featuring a well differentiated adenocarcinoma mixed with a transitional cell carcinoma (TCC) arising in the renal pelvis of a 63-year-old woman is presented. Daughter tumors, located in the ureter and the uretero-vesical junction, were entirely TCC in character. Immunohistochemical assessment of cell cycle-related proteins revealed overexpression of cyclin D1 but reduced p21WAF1/Cip1 or PCNA expression in the adenocarcinomatous regions. Conversely, expression of p21WAF1/Cip1 and PCNA was high in the TCC components. Immunohistochemical staining for p53 was negative and PCR-SSCP analyses confirmed the absence of any mutation. Therefore, assessments on the altered expression of cell cycle-related elements may contribute to our understanding of tumor biology in adenocarcinomas and TCCs of the renal pelvis and to identifying the similarities and differences between the two different cell types.      

Tumoricidal activity of high-dose tumor necrosis factor-alpha is mediated by macrophage-derived nitric oxide burst and permanent blood flow shutdown

This study investigates the role of tumor nitric oxide (NO) and vascular regulation in tumor ulceration following high-dose tumor necrosis factor-alpha (TNF) treatment. Using TNF-responsive (MethA) and nonresponsive (LL2) mouse tumors, tumor NO concentration was measured with an electrochemical sensor and tumor blood flow by Doppler ultrasound. Mice were also pretreated with a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. Tumors harvested from TNF-treated mice were cryosectioned and immunostained for murine macrophages, or/and iNOS. MethA tumor-bearing mice were depleted of macrophages. Pre- and post-TNF tumor NO levels were measured continuously, and mice were followed for gross tumor response. In MethA tumors, TNF caused a 96% response rate, and tumor NO concentration doubled. Tumor blood flow decreased to 3% of baseline by 4 hr and was sustained at 24 hr and 10 days post-TNF. Selective NO inhibition with 1400 W blocked NO rise and decreased response rate to 38%. MethA tumors showed tumor infiltration by macrophages post-TNF and the pattern of macrophage immunostaining overlapped with iNOS immunostaining. Depletion of macrophages inhibited tumor NO increase and response to TNF. LL2 tumors had a 0% response rate to TNF and exhibited no change in NO concentration. Blood flow decreased to 2% of baseline by 4 hr, recovered to 56% by 24 hr and increased to 232% by 10 days. LL2 tumors showed no infiltration by macrophages post-TNF. We conclude that TNF causes tumor infiltrating, macrophage-derived iNOS-mediated tumor NO rise and sustained tumor blood flow shutdown, resulting in tumor ulceration in the responsive tumor.     

喉癌癌变过程中CyclinE和p53表达的临床病理学意义

目的研究喉癌癌变过程中ＣｙｃｌｉｎＥ和ｐ５３表达的临床病理学意义。方法用免疫组化检测２０例喉正常粘膜、４０例不典型增生病变和６０例喉癌组织中ＣｙｃｌｉｎＥ和ｐ５３的表达。结果ＣｙｃｌｉｎＥ和ｐ５３阳性表达均定位于细胞核。在喉癌癌变过程中,喉正常粘膜、不典型增生病变和喉癌中ＣｙｃｌｉｎＥ阳性表达率分别为５．０％（１／２０）、２０．０％（８／４０）和４５．０％（２７／６０）（Ｐ＜０．００１）。ｐ５３阳性表达率分别为０、３０．０％（１２／４０）和６１．７％（３７／６０）（Ｐ＜０．００１）。淋巴结转移组ＣｙｃｌｉｎＥ阳性表达率明显高于非淋巴结转移组;ＣｙｃｌｉｎＥ和ｐ５３阳性表达显著性相关（Ｐ＜０．０５）。结论ＣｙｃｌｉｎＥ和ｐ５３异常表达可能是喉癌发生中早期分子事件,对喉癌发展起重要作用。     

Expression of nitric oxide synthase, cyclooxygenase, and p53 in different stages of human gastric cancer

The present study evaluated the significance of nitric oxide synthase (NOS), cyclooxygenase (COX) expression and p53 status in 55 patients with gastric adenocarcinoma and relationship of these molecular markers to tumor characteristics and metastatic potential. Immunohistochemical technique was used to identify the cellular location and distribution of the enzymes in the specific cells of gastric tumors. In gastric cancer tissue, the expression of inducible enzymes, iNOS and COX-2, increased significantly with increasing tumor stage (P=0.015, P=0.001, respectively), size (P=0.025, P=0.001, respectively) and the presence of metastases (P=0.002, P=0.015, respectively). The expression of constitutive enzymes, ecNOS and COX-1, followed the opposite pattern. COX-1 was significantly reduced in advanced gastric tumors (P=0.007) and tumors larger than 5 cm (P=0.007). Reduced expression of ecNOS was also observed in advanced gastric tumors; however, this did not reach statistical significance. 53% of gastric tumors showed accumulation of p53. This was significantly higher in advanced tumors (P=0.004), larger than 5 cm (P=0.015) with metastases (P<0.001). Gastric tumors positive for accumulation of p53 had significantly stronger expression of iNOS (P=0.018) and COX-2 (P=0.01) enzymes than tumors negative for this nucleophosphoprotein. We conclude, that tumor-associated nitric oxide production, as well as COX-2 overexpression, may promote gastric cancer progression by providing a selective growth advantage to tumor cells with non-functioning p53.     

Expression of endothelial and inducible nitric oxide synthase in benign and malignant lesions of the breast and measurement of nitric oxide using electron paramagnetic resonance spectroscopy

BACKGROUND: Nitric oxide (NO) is present in various human solid tumors and tumor cell lines, and it is believed that NO plays an important role in tumor growth. An increased NO concentration catalyzed by NO synthase (NOS) is cytotoxic and can promote apoptosis. The expression of endothelial NOS (e-NOS) and induced NOS (i-NOS) was examined in various breast tumors. METHODS: Immunohistochemical staining with a monoclonal antibody (Ab) against e-NOS and a polyclonal Ab against i-NOS was performed on paraffin embedded tissues from 41 benign breast lesions, 9 in situ breast lesions, and 54 invasive breast lesions. Functionality was confirmed by detection of NO using spin-trapping electron paramagnetic resonance (EPR) spectroscopy. RESULTS: e-NOS expression was found in 2 benign lesions (5%; 1 fibroadenoma and 1 proliferative mastopathy), in 5 in situ lesions (56%), and in 33 invasive lesions (61%). None of the benign lesions was positive for i-NOS, but 6 in situ lesions (67%) and 33 invasive lesions (61%) showed tumor cell staining. In particular, capillaries that were embedded in lymphocytic stroma showed a positive reaction for e-NOS. The functionality of NOS was demonstrated by direct NO formation using the EPR spin-trapping method. Tumors that were positive for e-NOS were observed more often in younger patients (P = 0.05). These tumors more frequently were highly differentiated or moderately differentiated and more often showed invasive ductal subtypes and a lower proliferation rate. Tumors that were positive for both e-NOS and i-NOS were more likely to be lymph node negative tumors. Both i-NOS-expressing lesions and e-NOS-expressing lesions showed strong coexpression (P = 0.00001). CONCLUSIONS: NOS is detected predominantly in in situ lesions and invasive breast lesions but rarely in benign lesions. NOS is found more frequently in invasive carcinomas with low malignancy. Using the spin-trapping EPR method, this study demonstrates direct NO formation in human breast tumors for the first time.     

Inducible nitric oxide synthase (iNOS) is expressed similarly in multiple aberrant crypt foci and colorectal tumors from the same patients

Aberrant crypt foci (ACF) are the earliest identified neoplastic lesions in the colon. Aberrant expression of inducible nitric oxide synthase (iNOS or NOS2) has been documented in colorectal tumors, but expression of iNOS has not been reported in human ACF or multiple neoplastic lesions from the same patient. Immunohistochemical expression of iNOS was evaluated in 42 ACF, 14 adenomas, and 25 carcinomas and their adjacent normal mucosa. iNOS was strongly expressed in the normal colonic epithelial cells of all patients; it was markedly reduced in 21 of 42 (50%) ACF and in 14 of 25 (56%) carcinomas. The expression of iNOS was remarkably similar in multiple lesions from the same patient (P < 0.0001). These results suggest that the reduced expression of iNOS is a very early event in the development of some human colorectal tumors, and that host factors control the expression of iNOS similarly in premalignant and malignant colonic epithelial cells.     

Reduced Fhit expression occurs in the early stage of esophageal tumorigenesis: no correlation with p53 expression and apoptosis

The FHIT gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene involved in multiple tumors, including esophageal carcinoma. We analyzed Fhit expression using an immunohistochemical method in invasive carcinoma, carcinoma in situ (CIS) and dysplasia, in paraffin sections of 75 esophageal squamous cell carcinomas (ESCs) to further elucidate the role of Fhit protein in esophageal carcinogenesis. In addition, we also examined whether Fhit expression correlated with p53 expression and apoptosis. Compared to adjacent normal mucosa, significant loss or reduction of Fhit expression was noted in 67 of 75 (89.3%) invasive ESCs, in 13 of 19 (68.4%) CIS lesions, and in 10 of 23 (43.5%) dysplastic lesions. There was a progressive loss or reduction of Fhit expression with progressive increases in the severity of histopathological changes (p < 0.001). However, there was no association between Fhit expression and clinicopathological findings, including tumor stage, lymph node metastasis, or overall survival. Moreover, Fhit expression was not significantly associated with p53 expression and apoptosis. These results indicate that abnormal Fhit expression is a common event in the early stage of ESC development and may occur independently of p53 expression and apoptosis mechanisms.     

Nitric oxide and ionizing radiation synergistically promote apoptosis and growth inhibition of cancer by activating p53

Nitric oxide (NO) is a potent tumor radiosensitizer; however, its clinical use is limited by systemic side effects. We have demonstrated previously that gene transfer of the human inducible NO synthase (iNOS) gene into tumor cells and tumors induces high-output NO production that significantly enhances tumor radioresponsiveness, with no observed side effects. Notably, iNOS gene transfer enhances tumor radioresponsiveness via apoptotic cell death. Because NO and ionizing radiation are both known to promote p53-dependent apoptosis, we hypothesized that p53 activation might be a primary mechanism for the synergy of these two genotoxic stresses. We report that NO and ionizing radiation synergistically activate p53 in colorectal cancers grown in athymic mice by augmenting phosphorylation of p53 at serine 15. The effect of NO and ionizing radiation on tumor cell apoptosis and tumor radioresponsiveness is significantly reduced in p53 knockout isogenic cancer cell lines. Furthermore, the transfer of both p53 and iNOS genes into tumor cells lacking functional p53 enhanced their radioresponsiveness more than transfer of either gene alone.     

Antioxidative response for nitric oxide production in breast carcinoma

Our aim was to study the role of oxidant and nitric oxide (NO)-derived damage in human breast carcinomas by studying the expression of nitrotyrosine in tumor tissue. To elucidate whether nitrotyrosine levels associate with NO synthesis and have an effect on antioxidative enzyme response or angiogenesis, we also studied the expression of all three nitric oxide synthases (NOS), manganese superoxide dismutase (MnSOD), catalase and vascular endothelial growth factor (VEGF) in the tumors. A large set of breast cancer samples in microarray blocks were stained with antibodies to nitrotyrosine, iNOS, eNOS, nNOS, MnSOD, catalase and VEGF. Nitrotyrosine expression was seen in 56% of the cases. There was a close relationship between the expression of nitrotyrosine and all three NOS isoforms (for all p<0.0005), catalase (p<0.0005) and MnSOD (p=0.043), in addition enlarged tumor size was in association with high nitrotyrosine (p=0.046), eNOS (p=0.005) and VEGF (p=0.046) levels. Our findings suggest that NO-derived oxidative damage takes place and its level associates to NOS synthesis in human breast cancer. The current results also imply the attempt of cells to hedge against effects of NO by increasing their MnSOD and catalase expression.