Sequential histochemical and morphometric studies on preneoplastic and neoplastic lesions induced in rat colon by 1 ,2-dimethylhydrazine

The sequential histochemical changes during colon carcinogenesiswere studied in male Sprague-Dawley rats given 16 weekly subcutaneousinjections of 15 mg 1,2-diinethyl- hydrazine per kg body wtand serially killed at regular in tervals. Cryostat sectionswere used to study the mucus content of the colonic mucosa withthe periodic acid Schiff's reaction, and enzyme histocheinicalmethods were applied to investigate the activity of some keyenzymes of carbohydrate metabolism at different stages of carcinogenesis.Enlarged mucus-rich crypts with a marked hypercellularlty (149%of control as determined morpbometrically) appearing very earlyduring carcinogenic treatment revealed almost normal activitiesof glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase(G6PDH) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Hyperbasophllic crypts lacking mucus production were observedlater and showed a loss of G6Pase, but marked increase of G6PDHand GAPDH activity. Mucus-rich signet ring cell carcinomas showedthe same enzymatic pattern as the mucus-rich crypts, whereasmucus-free adenocarcinomas and undifferentiated carcinomas revealeda loss of G6Pase and highly increased G6PDH and GAPDH activities.The results showed that focal changes in polysaccharide contentand in the activity of some enzymes of carbohydrate metabolism,as observed in various organs, also accompany the carcinogenicprocess in the colon. This supports the concept that aberrationsin carbohydrate metabolism play an important role during theprocess of carcinogenesis.     

K-ras mutations and mucin profile in preneoplastic lesions and colon tumors induced in rats by 1,2-dimethylhydrazine

K-ras and mucin profile variations, associated with intestinal carcinogenesis, were studied in the preneoplastic lesions, mucin-depleted foci (MDF) and aberrant crypt foci (ACF), and in colonic tumors induced in rats by 1,2-dimethylhydrazine (DMH). The frequency of lesions with K-ras mutations was 23% (3/13), 5.5% (1/18) and 100% (14/14) in MDF, tumors and ACF, respectively. Two of three MDF mutated in K-ras also carried a missense mutation in Apc. We also tested the expression of MUC2, a mucin abundantly expressed in normal colon and M1/MUCA5C, up-regulated in colon carcinogenesis, using immunohistochemistry. MDF and tumors showed a dramatic reduction in the expression of MUC2, whereas ACF showed only a slight reduction. The expression of M1/MUC5AC was almost absent in normal mucosa, but was increased in all the lesions (MDF, tumors and ACF). The expression of the intestinal trefoil factor (ITF), a marker of goblet cell lineage, was reduced in MDF and tumors compared to normal mucosa but not in ACF. In conclusion, although K-ras mutations are present in all ACF, they are less frequent in MDF and tumors; M1/MUC5AC is a marker associated with all preneoplastic events while the reduction of MUC2 and ITF expression is selectively associated with more advanced lesions such as MDF and tumors.     

MUC1、MUC2及MUC5AC在大肠黏液腺癌组织中的表达及意义

目的探讨黏蛋白MUC1、MUC2和MUC5AC在大肠黏液腺癌组织中的表达及意义。方法采用免疫组织化学法的SP法,检测40例大肠黏液腺癌组织中黏蛋白MUC1、MUC2和MUC5AC的表达。结果大肠黏液腺癌组织中MUC1、MUC2和MUC5AC的阳性表达率分别为55.0%、82.5%和77.5%。大肠黏液腺癌组织中MUC1、MUC2及MUC5AC的表达与肿瘤大小及分化程度无关(P>0.05),MUC1表达与浸润深度、淋巴结转移、Dukes分期呈负相关,而MUC2、MUC5AC表达与浸润深度、淋巴结转移、Dukes分期呈正相关(P<0.05)。结论MUC1、MUC2和MUC5AC的表达可预示大肠黏液腺癌的浸润转移潜能。     

MUC1, MUC2, MUC5AC, and MUC6 expressions in gastric carcinomas: their roles as prognostic indicators.

BACKGROUND: Although mucin expressions appear to be correlated with prognoses in patients with various cancers, several studies have reported conflicting and inconclusive results on the prognostic significance of mucin expression in gastric carcinomas. METHODS: To clarify the correlations between clinicopathologic profiles and the patients' survival, the expression of MUC1, MUC2, MUC5AC, MUC6 mucins and the p53 protein were evaluated immunohistochemically in 300 consecutive gastric carcinomas using the tissue-array method. In addition, 59 gastric adenomas and 57 adenoma-associated carcinomas were investigated. RESULTS: MUC1 was expressed in 2 (3.4%) cases of gastric adenoma, and MUC2 in 19 (32.2%) cases of gastric adenoma, out of a total of 59 lesions. In consecutive gastric carcinomas, 24.3% of gastric carcinomas expressed MUC1, 27.3% expressed MUC2, 38.0% expressed MUC5AC and 12.7% expressed MUC6. The rate of MUC1 expression in gastric carcinomas was significantly higher than in associated gastric adenomas (P < 0.01). The patients with MUC1-positive carcinomas showed significantly poorer survival than those with MUC1-negative carcinomas. On the other hand, MUC2, MUC5AC and MUC6 expressions were not significantly associated with patient survival. Interestingly, combined evaluation revealed that the group with the MUC1-negative plus p53-negative expression pattern showed a better prognosis than the remaining cases. In contrast, the group with the MUC2-negative plus p53-positive pattern showed a worse prognosis. CONCLUSIONS: Mucin expression is altered in gastric adenoma and carcinoma, and MUC1 mucin expression is significantly associated with poorer outcome in gastric carcinomas. A MUC1-negative plus p53-negative pattern or a MUC2-negative plus p53-positive pattern may predict outcome in patients with gastric carcinomas.     

Distribution of preneoplastic lesions and tumors, and beta-catenin gene mutations in colon carcinomas induced by 1,2-dimethylhydrazine plus dextran sulfate sodium

Mucin-depleted foci (MDF) are considered as useful biomarkers in rat colon carcinogenesis. The purpose of the present study was to examine the mechanism(s) underlying rat colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus 1% Dextran Sulfate Sodium (DSS). Twelve male F344 rats were given subcutaneous injections (40mg/kg body) of DMH twice a week. They received DSS in the drinking water for 1 week after the first injection of DMH and then were maintained on tap water. The rats were sacrificed at 10 and 14 weeks after the first injection of DMH. Colon tissues were divided into 10 segments from anus to cecum (A/J) and stained with Alcian blue (AB) to identify MDF. We found that MDF and tumors were induced in the rat colon after treatment with DMH plus DSS and that the number of MDF in each segment of the colon was significantly correlated with that of tumors (p=0.006). In addition, we found that the beta-catenin protein was accumulated in cytoplasm and nuclei of MDF and the frequent beta-catenin gene mutations in the colon tumors. These results suggest that MDF is closely related to rat colon carcinogenesis induced by DMH plus DSS.     

Inhibitory effect of dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine on two different categories of colon preneoplastic lesions induced by 1,2-dimethylhydrazine in F344 rats

Sphingolipids display a wide spectrum of biological activities, including cell growth, differentiation and apoptosis. However, precise mechanisms by which these compounds exert anticancer or cancer-preventive effects are not known. In the present study, we evaluated the preventive efficacy of enriched dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine (G(1)CM) on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and beta-catenin-accumulated crypt (BCAC) formation in F344 rats during initiation stage. We also examined whether G(1)CM affects cell proliferation and apoptosis in these lesions. Pure G(1)CM was isolated from rice bran. Forty-two rats were divided randomly into five experimental groups. Rats in groups 1-3 were given subcutaneous injections of DMH (40 mg/kg body weight) once a week for 2 weeks. One week before the first injection of DMH, rats in groups 2 and 3 were fed a diet containing 200 and 1,000 p.p.m. G(1)CM, respectively, for 5 weeks. Rats in group 4 were fed a diet containing 1,000 p.p.m. G(1)CM. Rats in group 5 were given the basal diet alone and served as untreated controls. The experiment was terminated 5 weeks after the start. Dietary G(1)CM at both doses (groups 2 and 3) significantly inhibited the induction of ACF and BCAC (P<0.001) when compared to group 1 treated with DMH alone. In groups 2 and 3, the proliferating cell nuclear antigen labeling indices of epithelial cells in ACF and BCAC were also lower than in group 1 (P<0.0001 for ACF, P<0.05 for BCAC). These results, that dietary G(1)CM has possible chemopreventive effects in the present short-term colon carcinogenesis bioassays, suggest that longer exposure may cause suppression of tumor development.     

Role of MUC1 and MUC5AC expressions as prognostic indicators in gastric carcinomas

BACKGROUND AND OBJECTIVES: The aim of this study is to clarify the relationship between the expression of MUC1 and MUC5AC mucins and the clinicopathological features in human gastric carcinomas using the mouse monoclonal antibodies VU-4H5 and Clone 45M1, respectively. Furthermore, the possibility of using phenotypes (MUC1+/MUC5AC+, MUC1+/MUC5AC-, MUC1-/MUC5AC-, MUC1-/MUC5AC+) to predict prognosis of the patients is evaluated. METHODS: Formalin-fixed, paraffin wax-embedded tissues from 76 cases of gastric cancer were examined for the expression of MUC1 and MUC5AC mucin antigens immunohistochemically using the avidin-biotin-peroxidase method. RESULTS: Of the 76 cases, MUC1 and MUC5AC immunoreactivities were observed in 49 (64.5%) and in 32 (42.1%) of gastric carcinoma tissues, respectively. MUC1 expression was significantly correlated to the depth of invasion, lymph node metastasis, peritoneal dissemination, and tumor stage. On the other hand, MUC5AC was inversely associated with depth of invasion, lymph node metastasis, liver metastasis, and tumor stage. Multivariate analyses indicated that tumor stage and MUC1 mucin expression were independently correlated with overall survival. The patients with MUC1+/MUC5AC- antigen staining in carcinoma tissues showed the lowest survival rate among four phenotypes. In contrast, the patients with MUC1-/MUC5AC+ antigen staining in carcinoma tissues showed the highest survival rate. CONCLUSIONS: Altogether these data suggest that combined evaluation of MUC1 and MUC5AC mucin staining may be clinically helpful to predict outcome in patients with gastric cancer.     

Bombesin enhances experimental carcinogenesis induced in rat colon by 1,2-dimethylhydrazine

The effects of bombesin on the colonic mucosa and on the incidence,number, size and histology of colon cancers induced by 1,2-dimethylhydrazine(DMH) were studied in Fischer 344 rats. In experiment 1, ratswere randomized into three groups to receive either saline orbombesin (10 or 30 µg/kg body wt) to determine the labelingindex of normal colonic mucosa. In experiment 2, rats were given20 weekly injections of DMH (20 µg/kg body wt) and receivedeither saline or bombesin (10 or 30 µg/kg body wt) everyother day for 24 weeks. Administration of bombesin significantlyincreased the labeling indices of colonic mucosa in a dose-dependentmanner. Chronic administration of bombesin at both dosages withDMH caused significant increases in the incidence, number anddepth of involvement of colon cancers; however, it did not affectthe size and histological type of colon cancers. In addition,bombesin at the dose of 30 µg/kg significantly increasedthe labeling index of colon cancer. These results suggest thatbombesin stimulates the cell proliferation of colonic mucosaand colon cancer and enhances colon carcinogenesis in rats.     

胃癌组织中黏蛋白MUC2和MUC5AC的表达及其临床意义

目的　研究黏蛋白 MUC2和 MUC5 AC在胃癌组织中的表达及其与临床病理参数之间的关系。方法　应用免疫组化 S- P法检测 74例胃癌组织中 MU C2和 MU C5 AC蛋白的表达。结果　胃癌组织中 MU C2和MU C5 AC蛋白阳性表达率分别为 5 1.4%和 40 .5 %。胃癌组织中 MU C2蛋白的表达与组织学类型、淋巴结转移和病理分期无关 (P>0 .0 5 ) ,仅与 L auren分型有关 (P<0 .0 5 ) ;而 MU C5 AC蛋白在胃癌中的表达与胃癌组织学类型和淋巴结转移无关 (P>0 .0 5 ) ,但与病理分期和 L auren分型密切相关 (P<0 .0 5 )。结论　 MU C2和 MUC5 AC黏蛋白在胃癌中的表达反映了胃癌细胞的不同方向分化 ,随着胃癌的进展 ,弥漫型胃癌可能向肠型胃癌分化     

Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression

Intestinal metaplasia is a well-established premalignant condition of the stomach that is characterized by mucin carbohydrate modifications defined by histochemical methods. The purpose of the present study was to see whether the expression of mucin core proteins was modified in the different types of intestinal metaplasia and to evaluate the putative usefulness of mucins as "molecular markers" in this setting. We used a panel of monoclonal antibodies with well-defined specificities to MUC1, MUC2, MUC5AC, and MUC6 to characterize the expression pattern of mucins. In contrast to normal gastric mucosa, the complete form or type I intestinal metaplasia (n = 20) displayed little or no expression of MUC1, MUC5AC, or MUC6 in the metaplastic cells and strong expression of the intestinal mucin MUC2 in the goblet cells of all cases. The incomplete forms of intestinal metaplasia, type II (n = 25) and type III (n = 16), expressed MUC1 and MUC5AC in every case, both in goblet and in columnar cells. MUC6 was also expressed in 16 cases of type II intestinal metaplasia and in 11 cases of type III intestinal metaplasia. The intestinal mucin MUC2 was expressed in every case of incomplete intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the different types of intestinal metaplasia allows the identification of two patterns: one defined by decreased levels of expression of "gastric" mucins (MUC1, MUC5AC, and MUC6) and expression of MUC2 intestinal mucin, which corresponds to type I intestinal metaplasia, and the other defined by coexpression of "gastric mucins" (MUC1, MUC5AC, and MUC6) together with the MUC2 mucin, encompassing types II and III intestinal metaplasia. Our results challenge the classical sequential pathway of intestinal metaplasia (from type I to type III via a type II intermediate step).     

Alterations of nuclear pores in preneoplastic and neoplastic rat liver lesions induced by 2-acetylaminofluorene

The nuclear pore density and area were measured on freeze-fracturednuclei of ACI/N rat liver altered foci, adenomas and carcinomasinduced by 2-acetylaminofluorene, and compared with those ofnormal hepatocytes. The pore density of nuclei from these preneoplasticand neoplastic lesions was significantly higher than that ofhepatocytes, but there was no difference between lesions. Thearea of nuclear pores of the focus cells did not differ fromnormal hepatocytes, whereas the areas of pores of adenoma andcarcinoma cells were increased. Moreover, the nuclear pore areaof carcinomas was significantly greater than that of adenomas.These results suggest that some changes may occur in nuclearpores in the progress of tumorigenesis.     

Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non‐repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia. Int. J. Cancer 80:210–218, 1999. Published 1999 Wiley‐Liss, Inc.     

Distribution of laminin and collagen type IV in rat colon tumors induced by 1,2-dimethylhydrazine

Immunofluorescent distribution of basement membrane components laminin and collagen type IV was studied in 51 rat colon tumors induced by 1, 2-dimethylhydrazine. In normal colonic mucosa, adenomas and carcinomas in situ continuous basement membranes were present, while in adenocarcinomas they were altered to different extents. An uncoordinated loss, or dissociation, of the two markers studied was found: the degree of collagen type IV loss was often much higher than that of laminin in the same tumor. These data suggest that a reliable determination of cancer invasion by monitoring basement membrane alteration requires the use of several basement membrane markers.     

Expression and localization of MUC1, MUC2, MUC5AC and small intestinal mucin antigen in pancreatic tumors

Alterations in the expression of mucin family members play an important role as well as alterations in oncogenes and onco-suppressor genes in carcinogenesis and progression of pancreatic cancer. We analyzed the expression and localization of MUC1, MUC2, MUC5AC and small intestinal mucin antigen (SIMA) in pancreatic tumors. MUC1 expression was observed in almost all samples, whereas MUC2 expression was not. MUC5AC expression was observed in 73.9% of the cancerous regions, 48.7% of the dysplastic regions and 72.0% of the hyperplastic regions but not in the normal pancreatic duct. SIMA expression was observed in 45.7% of cancerous regions, 17.9% of the dysplastic regions and 8.0% of the hyperplastic regions. Furthermore, stromal expression of MUC1, MUC5AC and SIMA was observed in 37.0%, 60.9% and 26.1% of the cancerous regions, respectively. Stromal expression of these mucins was not observed in the hyperplastic regions and normal pancreatic duct and was observed in only two dysplastic regions. The survival of pancreatic cancer patients with stromal expression of MUC1 or SIMA was worse than that of other patients (P=0.04). In conclusion, the localization of mucin expression, especially stromal expression of MUC1 or SIMA, might be a prognostic factor for patients with pancreatic cancer.     

Ultrastructural changes in chemically induced preneoplastic focal lesions in the rat liver: a stereological study

Ultrastructural changes were investigated and quantified, using a stereological approach, in early gamma-glutamyltranspeptidase (GGT)-positive focal lesions, induced in the rat liver by treatment with a single initiating dose of diethylnitrosamine (DENA) followed by promotion with phenobarbitone (PB) for 30 weeks. Within the extra-hepatocyte environment of focal tissue, the mean volume occupied by Ito cells was markedly decreased, whilst that occupied by endothelial and Kupffer cells was increased, when compared to uninvolved tissue from the same rat livers. The bile canaliculi were dilated, but no significant differences in the mean volume occupied by the sinusoidal and Disse spaces were noted. In focal hepatocytes there was a striking overproduction of lipid droplets and proliferation of smooth endoplasmic reticulum (sER). Whorls of concentrically arranged, parallel ER membranes were found only in the hepatocytes of preneoplastic foci, in association with the proliferated sER, and never in the surrounding, uninvolved tissue. The increase in mean volume of the sER, lipid droplet and cytoplasmic matrix compartments, together with the appearance of whorls, were the major contributing factors to the marked hypertrophy seen in focal hepatocytes. The mean volume of the rough endoplasmic reticulum, mitochondrial, lysosomal, peroxisomal and nuclear compartments per hepatocyte also increased, but contributed to a lesser extent to the cellular hypertrophy. It is speculated that whorls may be structural adaptations, resulting from a possible alteration in the normal feedback control of cholesterol synthesis, for the production of sterols and the biogenesis of sER in eosinophilic-type focal cells. The significance of changes observed in focal tissue, and the high biological variation noted between foci, is discussed in relation to the hepatocarcinogenic process.     

Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium

Background  

Helicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry.