Immunocytochemical detection of the onco-developmental protein oncomodulin in pre-neoplastic and neoplastic hepatocellular lesions during hepatocarcinogenesis in rats

Oncomodulin is a calcium-binding protein, detectable in extra-embryonic human and rat placental cells and in a wide variety of tumors, but not in any normal embryonic or adult rodent or human tissues. It is also absent from proliferatively active fetal or regenerating adult rat liver. The presence of this oncodevelopmental marker was investigated in pre-neoplastic and neoplastic liver lesions during hepatocarcinogenesis induced in rats by DENA treatment, using an antibody raised against purified oncomodulin. Positive immunostaining was observed in foci of altered hepatocytes, in neoplastic nodules and in HC, but not in the histologically normal surrounding liver parenchyma. The proportion of oncomodulin-positive foci gradually rose from 20-25% at 2-3 months after DENA treatment, to about 88% at 6 months and later. The proportion of positive neoplastic nodules increased from 50% at 5 months to about 73% (range 36-100) at 9 months and later; 88% of the HC found 10 to 20 months after DENA treatment were also positive. That early neoplastic nodules are oncomodulin-positive in a proportion (50%) similar to that of foci after the same duration of treatment is consistent with a lineage relationship between them but makes it unlikely that oncomodulin expression conditions the focus-nodule transition. The role, if any, of oncomodulin in malignant progression remains to be elucidated. It seems out of the question that it is a simple correlate of proliferative activity.     

Immunohistochemical expression of the C-fos protein in preneoplastic and neoplastic lesions during hepatocarcinogenesis in rats

We performed an immunohistochemical study of the c-FOS protein, in the preneoplastic and neoplastic liver lesions in rats. The majority of the lesions at all stages contained an average of 50 to 60 percent of c-FOS immunopositive nuclei: This contrasted with the non-involved parenchyma, which exhibited a low incidence of positivity (less than 10 percent). No relation with proliferation or apoptosis was observed in the lesions, whereas the c-FOS protein was overexpressed within a few hours of partial hepatectomy. Moreover, our results indicate that increased c-FOS protein content is insufficient to elicit full malignancy.     

Immunohistochemical study on oncogene product ras p 21 in thyroid carcinoma

The oncogene product ras p 21 was detected by an immunohistochemical procedure in 132 cases of thyroid carcinoma and 14 cases of normal thyroid. In addition, semiquantitative analysis using percentage of positive cells and a serially diluting test of the primary antibody were performed in papillary and follicular carcinoma, and its non-neoplastic counterparts. Papillary carcinoma showed identical results in the immunostaining and semiquantitative analysis between minute and advanced carcinoma. Papillary and follicular carcinoma exhibited significantly increased expression of ras p 21 in the cytoplasm, although the former showed much more enhanced expression of ras p 21 than that in the latter. The immunostaining for ras p 21 in papillary carcinoma differed clearly from non-neoplastic counterparts in which ras p 21 expression was moderately enhanced, although this difference was statistically disappeared between follicular carcinoma and its non-neoplastic counterparts. However, non-neoplastic tissue located close to these neoplasms showed more increased immunostaining for ras p 21 than that observed in the thyroid tissues remote from the neoplasm, and in normal thyroid. Squamous cell, anaplastic and medullary carcinoma demonstrated non or poor expression of ras p 21. These results suggested that ras p 21 had some effects on papillary and follicular carcinoma, and the effects of oncogene product on the neoplasm and non-neoplastic counterparts differed considerably depending upon a histological type of thyroid carcinoma.     

Immunohistochemical detection of the ras oncogene p21 product in an experimental tumour and in human colorectal neoplasms

The monoclonal antibody Y13 259 to the ras oncogene protein product p21 was used in an immunohistochemical study of ras expression in human colorectal neoplasms. The ability of the antibody to detect enhanced levels of ras expression was confirmed by its use with an experimental neoplasm known to express ras at high levels. Human colonic adenocarcinomas in general showed a similar staining intensity to that seen in normal mucosa. Adenomas however showed consistently high p21 expression as indicated by staining intensity. This suggests that elevated ras expression may be important in the development of adenomas, but that high levels need not be sustained in the conversion to invasive carcinoma.     

ras oncogene detection in pre-neoplastic lesions: possible applications for diagnosis and prevention.

The development of methodologies for the early detection of genetic mutations is an important issue in the prevention and treatment of malignancy. Current knowledge of the association of specific genetic alterations with the development of certain types of tumors enables us to approach the early detection of cancer long before histologic or pathologic evidence indicates the development of neoplastic tumor growth. The identification of genetic alterations as biological markers allows for early diagnosis, which in turn may dictate a particular regimen of treatment to prevent subsequent tumor development. This commentary presents an evaluation of the current status of procedures for the early detection of mutations in the ras oncogene family and their relevance to diagnosis and prevention of neoplastic disease.     

Immunohistochemical detection of c-myc and c-erbA products in diethylnitrosamine-induced preneoplastic and neoplastic liver lesions in rats

An immunohistochemical study of c-myc and c-erbA products (p-myc and p-erbA) in preneoplastic and neoplastic rat liver lesions showed that the longer the time of hepatocarcinogenic treatment, the higher the proportion of lesions, whatever their type, showing p-erbA positive cells (p-erbA+). The proportion of p-myc positive foci (the majority of which are also p-erbA+), which was low at the focus stage, increased at the nodule and tumor stages. The incidence of p-myc positivity (alone or combined with erbA positivity) in the nodules decreased from the nodule to the tumor stage. For tumors, three types of altered phenotypes were found, namely: p-myc+, p-erbA+ or p-myc+/p-erbA+. Nevertheless, there were regions in these tumors that were negative. At a given stage, all types of lesions exhibited about the same incidence of immunopositivity for the two oncogene products (expressed either alone or together), the presence of which did not correlate with proliferative activity. Since the fraction of lesions that undergo full malignant progression is much smaller than the proportion of lesions that express c-myc and/or c-erbA proteins, our data exclude the possibility that increased incidence of p-myc/ and/or p-erbA+ cells might be sufficient for inducing full malignancy. A significant proportion of foci and nodules, and of regions of these lesions and of tumors, were unlabeled, whatever the stage at which they are found. This indicates that, if implicated, the positive phenotype(s) would not be required for the maintenance of those liver alterations.     

Immunohistochemical study of oncogene product ras p21, c-myc and growth factor EGF in breast carcinomas.

The expression of the oncogene products ras p21, c-myc and the growth factor EGF (epidermal growth factor) was studied immunohistochemically in the tissue of 119 benign and malignant human breasts. In most cases, histologically normal breast tissues and benign lesions were found to be negative or poorly-expressive for reactivity with each antibody. Similar findings were observed in carcinoma in situ. Invading breast carcinomas demonstrated a significantly higher percentage of stained cells than that observed in benign lesions or carcinoma in situ; forty-two of 66 invasive breast carcinomas (63.6%) were highly-expressive for ras p21, thirty-eight (57.6%) for c-myc and twenty (30.3%) for EGF, but overall correlations between each oncogene expression and the clinical stage, tumor size or degree of differentiation were not found. The overall 5-year survival rate was studied in 58 patients with Stage II and III in association with each oncogene or EGF expression. Their survival rate was significantly effected by the EGF expression (0.05 less than p less than 0.1) but not by ras p21 or c-myc expression. Analysis of 36 specimens available with ER (estrogen-receptor) level revealed a significant correlation between the ER status and c-myc or E2 (estradiol) and a significant inverse correlation between ER status and ras p21 or EGF expression (P less than 0.05). The expression of ras p21, EGF and c-myc was not associated with metastatic tumor progression.     

Immunohistochemical demonstration of ras p21 oncogene product in normal, benign, and malignant human thyroid tissues

The p21 protein product of the cellular oncogene ras, designated ras p21, has been detected immunohistochemically in normal, benign and malignant human thyroid tissues. With the monoclonal antibody RAP-5 generated against a synthetic peptide corresponding to amino acid positions 10 to 17 of the ras p21 protein and an avidin-biotin-peroxidase complex (ABC), the expression of the ras p21 was evaluated in paraffin-embedded sections. Western blot analysis using fresh thyroid carcinoma tissue revealed double protein bands, one band was at molecular weight 21,000 and the other was a more rapidly migrating band at the molecular weight 17,500. Immunohistochemically, papillary adenocarcinomas of the thyroid showed moderate to intense stainings for ras p21 in most cases. Cytoplasmic and apical surface stainings were the most common patterns of immunoreactivity. Adenomas showed variable ras p21 positivity in cytoplasm and apical surface stainings of adenomas were negative to borderline in most cases. The cytoplasm of tissues of Hashimoto's thyroiditis, Graves' disease, and normal thyroid tissues was uniformly ras p21 positive, but the apical cell surface was nonreactive for ras p21 in all tissues. Judging from the findings obtained on this large series of normal, benign, and malignant thyroid tissues, the elevation of ras p21 may be a common event in thyroid neoplasm, and especially elevated ras expression in the apical cell surface may be characteristic to papillary carcinomas of the thyroid. This suggests that apical surface expression of ras p21 may be important in the development of thyroid carcinomas and be useful in differentiation of papillary adenocarcinoma.     

Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry

The rapid identification of the expression of oncogene products in specific cell types could be useful to investigate normal and malignant cell proliferation. We have developed a sensitive fixation - permeabilization technique (with 70% ethanol and 0.01% Triton x 100) for the detection of p21 ras oncoprotein and DNA content. Cell suspensions with negligible cell clumping, bright specific immunofluorescent staining were obtained with this method. Bivariate flow cytometry was then used to quantitate simultaneously the distribution of anti p21 ras oncoprotein (with a specific FITC-labeled antibody) and of total DNA (with propidium iodide). The study was carried out in human leukemic cell lines HL-60 and K562, human breast carcinoma cell line MCF-7 and fresh neoplastic cells from human acute leukemia. The p21 ras oncoprotein was found in all phases of cell cycle. The degree of its expression, however, varied widely in diploid (C0/G1) cells from different samples, which could be related to differences in the relative proportion of G0 and G1 cells. Compared to the conventional gel electrophoretic technique, the use of bivariate FCM is feasible, fast, requires fewer cells per sample (2 x 10(6] and allows both the ras oncogene expression in intact cell populations as well as its relationship with cell cycle phases to be studied.     

p21和IGF-II在肝癌、肝硬化组织中表达的研究

目的　探讨 p2 1、IGF II在肝硬化、肝细胞癌 (HCC)中表达的意义及其与肝细胞不典型增生(LCD)的关系。方法　用免疫组化S P法检测单纯肝硬化、癌旁肝硬化及肝细胞癌 (HCC) p2 1、IGF II表达情况。结果　p2 1、IGF II在癌旁肝硬化和单纯肝硬化组织中的阳性表达率 (92 .86 %和 75 % ;6 8%和 74 % )与HCC(5 4 .5 5 %和 36 .36 % )相比差别有显著性意义 (P <0 .0 5 )。IGF II与p2 1阳性表达存在相关关系。HBsAg阳性或伴LCD改变的肝硬化 ,p2 1、IGF II阳性率均高于HBsAg阴性或不伴有LCD改变的肝硬化 (P <0 .0 5 )。结论　 (1)在肝细胞癌变演化过程中 p2 1、IGF II可能发挥协同作用。 (2 )LCD可能是一群具有肿瘤增殖潜能的癌前细胞群 ,尤其有 p2 1、IGF II共同过度表达者 ,有发生HCC的高度危险。     

The H-ras oncogene product p21 and prognosis in human breast cancer

Summary The protein product of the H-ras oncogene, p21, has been measured semiquantitatively in solubilized particulate fractions of 160 primary tumours from patients presenting without evidence of distant metastatic breast cancer. Levels of p21 have then been related to factors of established prognostic significance, and to clinical outcome after primary treatment in terms of disease-free interval and survival times. p21 was detected by Western blotting in all tumour fractions, but amounts varied markedly between different tumours. There was no significant relationship between levels of p21 and the menopausal status of the patient, tumour oestrogen receptors, grade, and clinical stage. However, there was a significant trend for tumours to be associated with lymph node involvement as p21 was increasingly expressed. Elevated levels of p21 were also significantly related to early disease recurrence and death from cancer. Multivariate stepwise analysis showed that both p21 and lymph node status were independent statistically significant factors for disease recurrence and survival, and that no other parameter was significant for clinical outcome after adjustment for p21 and lymph node status. These results indicate that tumour levels of p21 are an important prognostic variable in patients with early breast cancer.     

Development of hydroxysteroid sulfotransferase-deficient lesions during hepatocarcinogenesis in rats

Rat liver cytosolic hydroxysteroid sulfotransferases form highlyreactive sulfurk acid esters from some benzylic alcohols, suchas 1-hydroxymethylpyrene. In this study we examined the expressionof hydroxysteroid sulfotransferase a (STa) in carcinogen-inducedenzyme-altered, presumably preneoplastic, rat liver foci. FemaleWistar rats were given a single i.p. injection of diethylnitrosamine(0.15 µmol/g body wt) 1 day after birth to induce theliver foci. After weaning, rats were given 1-hydroxymethylpyreneor phenobarbital continuously in their diet (250 or 500 p.p.m.respectively) for a total of 120 days. Carcinogen-induced liverfoci were identified by a change in the marker enzyme adenosinetriphosphatase. Immunohistochemkal staining of consecutive sectionsusing an anti-STa rabbit antibody demonstrated that STa wasexpressed at decreased levels in most of the adenosine triphosphatase-negativeliver foci. This effect was observed in both 1-hydroxymethylpyrene-and phenobarbital-treated animals. The decrease in STa contentin enzyme-altered foci may lead to a selective advantage ofthe preneoplastic cells in the presence of agents that are ableto form reactive sulfuric acid esters, such as 1-hydroxymethylpyrene.In some diethyl-nitrosamine/phenobarbital-treated rats, a smallnumber of atypical foci were observed, most of them showingenhanced expression of STa and unchanged to moderately increasedATPase activity.     

p21和IGF-Ⅱ在肝癌、肝硬化组织中表达的研究

目的探讨p21、IGF-Ⅱ在肝硬化、肝细胞癌(HCC)中表达的意义及其与肝细胞不典型增生(LCD)的关系.方法用免疫组化S-P法检测单纯肝硬化、癌旁肝硬化及肝细胞癌(HCC)p21、IGF-Ⅱ表达情况.结果 p21、IGF-Ⅱ在癌旁肝硬化和单纯肝硬化组织中的阳性表达率(92.86%和75%;68%和74%)与HCC(54.55%和36.36%)相比差别有显著性意义(P<0.05). IGF-Ⅱ与p21阳性表达存在相关关系. HBsAg阳性或伴LCD改变的肝硬化,p21、IGF-II阳性率均高于HBsAg阴性或不伴有LCD改变的肝硬化(P<0.05).结论 (1)在肝细胞癌变演化过程中p21、IGF-Ⅱ可能发挥协同作用.(2)LCD 可能是一群具有肿瘤增殖潜能的癌前细胞群,尤其有p21、IGF-Ⅱ共同过度表达者,有发生HCC的高度危险.     

H-ras p21 and peanut lectin immunoreactivity of hyperplastic, preneoplastic and neoplastic urinary bladder lesions in rats

Hyperplastic, preneoplastic and neoplastic urinary bladder lesions induced by bladder carcinogens and toxins in the rat were evaluated for immunoreactivity with polyclonal or monoclonal antibodies to H-ras p21 or binding to peanut lectin with avidin-biotin immunocytochemistry. A low proportion (less than 20%) of hyperplastic and neoplastic bladder lesions induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine and fixed in Bouin's fixative only were immunoreactive on the cell membrane with the antibodies to H-ras p21. Lectin binding was found for these lesions, as well, even in formalin-fixed tissue and for lesions induced by other carcinogens, but not in regenerative bladder hyperplasias after cyclophosphamide exposure or in bladder exposed to bladder tumor promoters. The latter lesions were also not immunoreactive with antibodies to p21. Our results suggest that this relatively simple technique might be used for identification and screening of tumors for involvement of ras oncogenes and carcinogen initiation.     

卵巢癌ras癌基因产物p21的定量分析

目的：探讨卵巢癌ｒａｓｐ２１表达与ＤＮＡ倍体水平及临床病理参数的关系。方法：采用流式免疫荧光技术。结果：３６例卵巢癌１４例ｒａｓｐ２１呈阳性表达，ｒａｓｐ２１阳性表达与组织分级无关：ｒａｓｐ２１阳性表达量在二倍体与异倍体之间有显著性差异；ｒａｓｐ２１阳性表达多见于粘液性卵巢癌。结论：ｒａｓｐ２１表达量与ＤＮＡ倍体相关     

Dual digital video-autofluorescence imaging for detection of pre-neoplastic lesions

AIM: The incorporation of autofluorescence (AF) to white light bronchoscopy has led to improved sensitivity for the detection of pre-neoplastic lesions in the airways. However, AF has difficulty distinguishing benign epithelial changes such as bronchitis, previous biopsy, and airway fibrosis from pre-invasive lesions, which necessitates extensive biopsy. This frequently results in longer procedural time and need for additional sedation that may compromise patient safety, increase the risk of bronchospasm, and bleeding from multiple endobronchial biopsies. We postulate that dual imaging with simultaneous video and AF bronchoscopy of the tracheobronchial tree could improve the low specificity observed with AF in the detection of pre-invasive lesions, leading to targeted biopsy, good correlation with pathological diagnosis and shorter procedural time. METHODS: Forty-eight patients with known or suspected of lung cancer underwent video and AF bronchoscopy, which were provided as real-time dual images with SAFE 3000 (Pentax, Tokyo) between March and August 2006. Biopsy specimens were taken from all suspicious areas with two random specimens from normal areas. Values were expressed as median and range, and p<0.05 was considered statistically significant. RESULTS: Twenty-five suspicious sites were detected by dual imaging bronchoscopy, and 126 endobronchial biopsies were evaluated, of which 22 (17.5%) were graded as moderate dysplasia and worse. Sensitivity and specificity of dual imaging for the detection of high-grade dysplasia were 86% and 94%, respectively, with good correlation between bronchoscopic assessment and pathology (r=0.77, p<0.0001). However, there were three random biopsy specimens obtained from normal or abnormal sites that showed severe dysplasia in two and moderate dysplasia in one. Median time taken for airway examination was 4 min (range, 4-4.8), and 5 min (range, 4-5) for biopsy, giving a total procedural time of 9 min (range, 8-10). There were no procedure-related complications noted. CONCLUSION: Dual imaging that allows simultaneous real-time assessment of the lesion with video and AF bronchoscopy not only achieves satisfactory sensitivity for the detection of pre-neoplastic lesions, importantly it improves specificity by allowing targeted biopsy, which has led to a marked decrease in procedural time and better patient safety.     

Development of cytochrome P-450-altered preneoplastic and neoplastic lesions during nitrosamine-induced hepatocarcinogenesis in the rat

The expression of four cytochrome (cyt.) P-450 isoenzymes has been studied in preneoplastic and neoplastic lesions during the course of nitrosamine-induced hepatocarcinogenesis in the female Wistar rat. Following exposure to diethylnitrosamine (50 or 100 ppm in the drinking water) for 10 days, animals were taken sequentially, and the livers were analyzed for the evolution of adenosine triphosphatase deficient focal lesions. These lesions were subdivided into different phenotypes with regard to their cyt. P-450 isoenzyme expression using serial frozen sections. Our results demonstrate that about 40% of the adenosine triphosphatase-deficient lesions show concomitant alterations in their cyt. P-450 isoenzyme contents. Of these lesions, islets which are characterized by decreased levels of at least three cyt. P-450 isoenzymes show a dramatic increase in their volumetric fraction of liver tissue with progression of time. Although only very few lesions express this phenotype, the contribution to the volumetric fraction of islet tissue raises from about 2% at 10 weeks to about 60% at 35 weeks after cessation of diethylnitrosamine treatment. By contrast, lesions which express less than two alterations in cyt. P-450 isoenzyme levels develop relatively slowly. Similar results were obtained when animals were exposed continuously to diethylnitrosamine for a period of up to 8 weeks. Following treatment of islet-bearing animals with phenobarbital, an induction of cyt. P-450 isoenzymes and NADPH-cyt. P-450-reductase was observed within preneoplastic and neoplastic lesions. This induction was most pronounced in large, expansively growing nodules, a type of lesion which displayed decreased levels of these enzymes in livers of animals not treated with phenobarbital. The elevation of the cyt. P-450 isoenzymes disappeared within 2 to 3 weeks after cessation of inducer treatment. Our results indicate that a high proportion of rapidly growing lesions has assumed a constitutive deficiency in cyt. P-450 isoenzyme expression during nitrosamine-induced hepatocarcinogenesis. This deficiency, however, is not an irreversible quality, since individual cyt. P-450 isoenzymes can be markedly induced by treatment with an enzyme inducer like phenobarbital. Thus, the observed decrease in cyt. P-450 expression during development of malignancy does not result from alterations in the cyt. P-450 encoding structural genes but may rather be related to abnormalities in the function of regulatory systems of a higher order which may play a central role in the maintenance of cell homeostasis.     

Immunohistochemical study of ras p21 expression in human gastric cancers and benign lesions

Expression of ras p21 in human gastric cancers, benign lesions and normal tissues was immunohistochemically evaluated by the avidin-biotin peroxidase complex (ABC) method with anti-ras p21 monoclonal antibody rp-28. Positive p21 immunoreactivity was shown in 23 (77%) of 30 gastric cancers, in 13 (48%) of 27 benign lesions and in 10 (22%) of 46 normal mucosa cases. Among them, strong staining was demonstrated only in 11 (37%) of 30 gastric cancers, but not in benign lesions and normal tissues. Cases showing more than 20% positive cell ratio were observed in 22 (73%) gastric cancers, in 11 (41%) benign lesions and 6 (13%) normal mucosa cases. Further, intracellular distribution of ras p21 is heterogeneous in gastric cancers, while it is homogeneous in benign lesions or normal tissues. The ABC method with rp-28 could be helpful for clinical differential diagnosis between gastric cancers and benign lesions by investigating three factors: staining intensity, positive cell ratio and intracellular distribution of ras p21.     

HIV and pre-neoplastic and neoplastic lesions of the cervix in South Africa: a case-control study

Background  

Cervical cancer and infection with human immunodeficiency virus (HIV) are both major public health problems in South Africa. The aim of this study was to determine the risk of cervical pre-cancer and cancer among HIV positive women in South Africa.     

鼠实验性肝癌超微结构和ras p21免疫电镜观察

目的：动态观察二乙基亚硝胺诱发大鼠肝细胞肝癌过程超微结构和ｒａｓｐ２１免疫电镜改变。方法：对６０只二乙基亚硝胺诱癌的大鼠分期处死,进行电镜及ｒａｓｐ２１免疫电镜观察。结果：早期即见肝细胞内线粒体、粗面内质网、滑面内质网和核、核仁等显示一系列损伤性变化,小肝细胞灶性增生,并向嗜碱细胞和异常肝细胞转化,到诱癌中期以至诱癌晚期,异型肝细胞逐渐向癌细胞转化,并逐渐形成了癌细胞的超微结构特点。ｒａｓｐ２１免疫电镜观察发现,ｐ２１阳性颗粒除沿细胞膜分布外,还可见到内质网上有免疫反应物沉积,甚至在核膜上也观察到略弱的ｐ２１免疫复合物沉积。结论：随诱癌时间的延长,肝细胞逐渐向癌细胞转化,ｒａｓｐ２１阳性颗粒可在癌变细胞内多处分布。