Epidermal growth factor receptors in colorectal carcinoma

Nineteen samples of primary colorectal carcinoma and adjacent mucosa were examined for EGFr expression using radioligand binding assays and immunohistochemical staining with the monoclonal antibody EGFR1. Radioligand binding experiments showed expression of EGFr in both tumour and mucosa in all cases. In tumour samples EGFr levels ranged between 4 and 79 fmole per mg membrane protein (Kd = 0.1-0.4 X 10(-9) M). There was no significant difference in the level of EGFr expression between tumour and mucosa overall. Immunohistochemical staining with the EGFR1 antibody was useful in localising EGFr to epithelial elements although it was less sensitive than ligand binding assays.     

Expression of alpha, mu and pi class glutathione S-transferases in oval and ductal cells in liver of rats placed on a choline-deficient, ethionine-supplemented diet.

Expression of the alpha, mu and pi class glutathione S-transferases (GSTs) in hepatocytes, oval cells and ductal cells derived from the livers of rats placed on a choline-deficient, ethionine-supplemented (CDE) diet for 5 weeks was investigated. An overall decrease in the expression of alpha and mu class GSTs and an over-expression of pi class GST was observed in the liver after CDE treatment as indicated by Northern blotting analysis. Massive disruption of the liver with oval cell infiltration in the sinusoids throughout the lobule occurred after 5 weeks CDE treatment. 'Duct-like' structures consisting of oval-like cells (ductal cells) with rounder nuclei and more cytoplasm than oval cells within the sinusoids were also apparent. Immunocytochemical analysis revealed that the altered expression of GST in the whole liver is attributed to a differential expression of alpha, mu and pi class GSTs in the different cell types in the liver, including hepatocytes, oval cells around the portal region and among the sinusoids, and oval-like cells (ductal cells) in the 'duct-like' structures. In vitro studies using purified oval-ductal cells and hepatocyte populations confirmed the differential expression of GSTs in the varying cell populations in situ. The expression of the alpha and mu class GSTs in hepatocytes does not appear to be altered by the CDE diet. Heterogeneity in distribution of pi class GST was observed in the hepatocyte population, some hepatocytes were stained strongly while no staining was observed in others. Oval and ductal cells represent two distinct populations displaying different expression of GSTs. Pi class GST was detected in the majority of oval and ductal cells. Alpha class GST was detected in < 5% of the oval cell population and was found in > 50% of the ductal cell population. In contrast, mu class GST was absent in ductal cells and was present in 24% of oval cells around the portal region. This supports the view that ductal cells are not of bile ductal origin since mu GST is present in normal bile duct epithelial cells. Furthermore the change in expression of GSTs in the liver after CDE treatment is attributed to the large increase in oval and ductal cell populations.     

Glutathione S-transferase expression in fetal kidney and Wilms' tumour

The glutathione S-transferases (GSTs) have been implicated in carcinogenesis and tumour drug-therapy resistance. In this study GST pi was the predominant isoenzyme in the fetal human kidney. It was present in differentiated epithelial structures but never in the primitive mesenchyme. By contrast most cases of Wilms' tumours showed GST pi in both epithelial structures and undifferentiated blastema. The level of expression, as assessed by immunostaining, was no more than moderate, and was generally higher in differentiated elements. In only one case was GST alpha found in Wilms' tumour. This study had demonstrated a difference between fetal kidney and Wilms' tumour blastema in terms of GST expression.     

Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system

Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 microM) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 microM), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.     

Decreased expression of the glutathione S-transferases alpha and pi genes in human renal cell carcinoma

Using the polymerase chain reaction (PCR), a human kidney glutathione S-transferase (GST) alpha cDNA clone (GST alpha 12 K) was synthesized; it is identical to a known liver GST alpha cDNA clone except for one base change (G----A), indicating that an alpha class gene expressed in human kidney is similar to one expressed in human liver. Comparisons were made in the expression of GST alpha and GST pi between renal cell carcinoma and adjacent non-neoplastic tissue. Messenger RNA expression in 30 cases was determined by Northern blotting, and GST protein from nine of these cases was analyzed by HPLC. The GST alpha gene products were expressed at near-zero levels. The GST pi gene product was the predominant GST in tumors, but was decreased in absolute amount compared with control tissue, the tumor/control ratios for the GST pi gene obtained by Northern blots and HPLC analysis being 0.50 +/- 0.07 and 0.36 +/- 0.07 respectively. The resulting pattern in renal cell carcinoma therefore shows a predominance of GST pi. Since it is assumed that renal cell carcinoma derives from the proximal tubular epithelial cells which are high in GST alpha, this implies a dedifferentation in the GST expression pattern.     

Glutathione transferase isoenzymes in normal and neoplastic human kidney tissue.

Glutathione transferase (GST) activity in the cytosolic fractions of renal cortex tumour was found to be significantly lower (215 +/- 156 mU/mg) than that present in the corresponding non-tumour (466 +/- 278 mU/mg) tissues. Using the immunoblotting technique, glutathione transferase isoenzymes expression in both tumour and non-tumour kidney was investigated. Alpha and pi class glutathione transferases were the most abundant enzymes in non-tumour kidney and were expressed by all samples investigated. Immunofluorescence analysis indicated that the pi class enzymes are localized mainly in the distal convoluted tubules, whereas alpha class enzymes are localized in the proximal tubules. In the tumour moiety the alpha class GST appears to be absent or expressed at low level as compared with non-tumour samples. On the contrary, no significant differences in the expression of pi class GST were found in tumour as compared with non-tumour tissues. Mu class GST protein was detected in 12 of 26 samples tested. When present, mu class GST constitutes a few per cent of total GST protein. Immunofluorescence studies indicate that mu class GSTs are localized within the distal convoluted tubules. According to the electrophoretic mobility at least two different mu GST subunits (26.5 and 27.5 kd) were found. In one sample only the faster mu class GST subunit was present, two samples expressed both types of GST subunits, whereas nine samples expressed only the slower GST subunit. With the exception of one sample, a reduction of mu class GST expression was seen in tumour as compared with non-tumour tissues. The decrease of activity seen in the cytosolic fraction of tumour kidney must be ascribed mainly to a reduction or to a lack of expression of alpha class GST and to a lesser extent of mu class GST.     

Alpha-methylacyl-CoA-racemase expression in adenocarcinoma, dysplasia and non-neoplastic epithelium of the stomach

Alpha-methylacyl-CoA-racemase (AMACR) is an essential enzyme in the oxidation of bile acid intermediates and branched-chain fatty acids. This study aims to examine the expression pattern, as well as diagnostic and prognostic significance, of AMACR in carcinoma, dysplasia and non-neoplastic epithelium of the stomach. A total of 158 cases, including 66 cases of gastric carcinoma (GC), 48 cases of dysplasia and 44 cases of non-neoplastic gastric mucosa, were examined by immunohistochemistry for AMACR. AMACR expression was divided into two categories: negative (negative-weak staining intensity) and positive (moderate-strong staining intensity). AMACR immunoreactivity was detected in only 2 of 44 (4.5%) cases of non-neoplastic epithelium. A significantly high frequency of AMACR expression was found in 40 of 48 (83.3%) cases of dysplasia and 34 of 66 (51.5%) carcinoma cases compared with cases of non-neoplastic epithelium (p < 0.05). The frequency of AMACR expression was significantly higher in dysplasia than in carcinoma cases (p < 0.05). AMACR expression was higher in intestinal- than diffuse-type GC (p < 0.05). In conclusion, this study suggests that AMACR immunostaining aids in distinguishing malignant or precancerous lesions from reactive epithelial atypia in gastric biopsy specimens. It also suggests that AMACR expression is more likely to be associated with intestinal-type adenocarcinoma in gastric carcinogenesis.     

Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation

Calreticulin is an endoplasmic reticulum luminal calcium-binding chaperone involved in various cellular functions and is a ligand for the scavenger receptor CD91. Recent studies, based on proteomic approaches on whole tissue samples containing both neoplastic and non-neoplastic cells, have shown alterations of Calreticulin expression in colon carcinomas, albeit with divergent results. The aims of this study were: 1) to assess the expression of Calreticulin and its receptor CD91 in 58 human colon adenocarcinomas, compared with paired normal mucosa, using a semi-quantitative immunohistochemical analysis, and 2) to examine associations between the tumour phenotypic features, and Calreticulin and/or CD91 expressions. Calreticulin expression was down-regulated in 51.7% human colon adenocarcinomas. Accordingly, quantitative immunoblot analysis showed that Calreticulin expression was significantly lower in human colonic cancer cell lines than in preparations of isolated human normal colonic epithelial cells. CD91 was co-expressed with Calreticulin in both normal colonic epithelial cells and pericryptic myofibroblasts. Calreticulin and CD91, that characterize the 'amateur phagocyte' function of epithelial cells, were both down-regulated in 48% of adenocarcinomas. Finally, Calreticulin expression was significantly associated with the mucinous differentiation of the tumour. Collectively, these results show that Calreticulin is likely to play a pivotal role in the differentiation of human colonic adenocarcinomas.     

TGF-beta expression in the human colon: differential immunostaining along crypt epithelium.

Samples of colorectal carcinoma, adenoma and normal colorectal mucosa were examined for the expression of TGF-beta by immunohistochemistry. Immunoreactivity for TGF-beta was present in 52 out of a total of 58 samples of normal mucosa examined. In adenomas and carcinomas TGF-beta expression was observed in eight out of ten and 46 out of 48 samples respectively and was largely restricted to epithelial cells. In normal mucosa differential expression of TGF-beta was present within epithelial cells, those in the upper parts of the crypts showing enhanced immunoreactivity compared to cells in the proliferative compartment. This pattern of differential staining is consistent with TGF-beta having an important role in the control of growth and differentiation in colonic mucosa.     

Expression of glutathione S-transferases and cytochrome P450 in normal and tumor breast tissue

The level of expression of glutathione S-transferases (GSTs) and cytochrome P450s in breast tissue are potentially important determinants in both the susceptibility of this tissue to the mutagenic effects of chemical carcinogens and in the response of breast tumors to chemotherapy. In this study we have investigated the expression of these proteins in 41 tumor and surrounding normal breast tissue samples by measurement of substrate metabolism. Western blot analysis and immunohistochemistry. In addition, we have quantitated the concentration of alpha, mu and pi class GST subunits using radioimmunoassay. All three classes of GST were expressed in breast tissue. The pi and mu class enzymes preponderate. Both the polymorphic mu class GST as well as a further form, present in all individuals, were found in high concentration. The polymorphic mu class GST was expressed in approximately 50% of the samples, which is consistent with the frequency of this polymorphism in the population and therefore does not appear to be a factor in susceptibility to this disease. Interestingly, although levels of the alpha class GST were very low, in two tumor samples extremely high levels of the B1B1 subunit were detected. Immunohistochemical studies showed significant variability in the localization of the pi class of GST between normal epithelial cells, infiltrating plasma cells and tumor cells, and in some samples GST pi appeared to be almost absent from the tumor tissue. No direct, or inverse correlation was found between GST pi concentration determined by radioimmunoassay and estrogen receptor levels. However, when studied by immunohistochemistry estrogen receptor negative tumors did tend to have higher GST pi content. The only cytochrome P450 detectable by Western blot analysis was a member of the P450IIC gene family. This was apparently distinct from the P450IIC proteins expressed in the liver and was detected in normal and tumor tissues to a similar extent.     

Glutathione content and glutathione-S-transferase expression in 1,3-bis(2-chloroethyl)-1-nitrosourea-resistant human malignant astrocytoma cell lines

gamma-L-glutamyl-L-cysteinylglycine (GSH) has been shown to inactivate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and quench DNA crosslink precursors of BCNU. Because of the central role of 2-chloroethyl-nitrosoureas in brain tumor chemotherapy, we investigated the intracellular GSH content and the expression of specific glutathione-S-transferases (GSTs) in three human malignant astrocytoma cell lines (UWR1, UWR2, and UWR3) of varying BCNU resistance to determine the interrelationship of these parameters with brain tumor BCNU resistance. GSH was assayed by ion-exchange high performance liquid chromatography after derivatization with 1-fluoro-2,4 dinitrobenzene. Both bulk and specific GST (acid, near-neutral, and basic) activities were examined using substrates that show high specificities to the different GSTs. Western blot analyses with antisera against GST-alpha, -mu, and -tau subunits were also performed on partially purified GST from the cells of each cell line. The results showed GSH content of 91, 46.5, and 28.3 nmol GSH/mg protein for UWR1, UWR2, and UWR3, respectively. Bulk GST activity (with 1-chloro-2,4-dinitrobenzene as substrate) also correlated with increasing BCNU resistance. Of the three GST classes examined by both substrate specificities and Western blotting, only the expression of the acidic form, GST-tau, correlated significantly with the rank order of BCNU resistance of the cell lines. GST-mu and -alpha were present in only trace amounts in all three cell lines.     

The relationship between tumour glutathione concentration, glutathione S-transferase isoenzyme expression and response to single agent carboplatin in epithelial ovarian cancer patients.

There is evidence to suggest that glutathione (GSH) and glutathione-S-transferases (GST) are important factors in determining sensitivity to cytotoxic drugs in vitro and in preclinical in vivo model systems. To define the relationship between tumour GSH concentration, GST isoenzyme expression and response to carboplatin in epithelial ovarian cancer (EOC), tumour samples from 39 patients with assessable disease after primary surgery were analyzed for GSH content and GST expression. Response was assessed after completing six courses of single agent carboplatin therapy. GSH was measured by high performance liquid chromatography (HPLC) in fresh tumour samples taken at primary laparatomy. GST isoenzyme expression was assessed by immunohistochemistry of fixed tumour material using antibodies specific for pi, alpha and mu classes. GST isoenzyme expression was defined as positive if the staining intensity was strong and more than 10% of tumour cells were involved. The mean GSH concentrations were: 8351 +/- 4496, 7211 +/- 5026, 6559 +/- 4573 and 3758 +/- 1885 (nmol g-1 tissue dry weight mean +/- s.d.) for tumours from patients who subsequently achieved a complete response (CR, n = 18), partial response (PR, n = 10) or who had static disease (SD, n = 7) or progressive disease (PD, n = 4) respectively. There was no relationship between GSH concentration and response (ANOVA, P = 0.32). There were also no relationship between GST isoenzyme expression and response (P Fisher''s exact test 0.51-0.55 and chi-squared test 0.98-0.99). In conclusion, there was no association between the concentration of GSH or expression of GST isoenzymes and response to single agent carboplatin in primary previously untreated EOC.     

Loss of glutathione S-transferases in pollution-associated liver neoplasms in white suckers (Catostomus commersoni) from Lake Ontario.

White suckers (Catostomus commersoni) are one of two species of bottom-feeding fish in which various liver neoplasms are more prevalent in urban/industrial sites in western Lake Ontario than in less polluted sites in the Great Lakes. Previous studies indicate that white suckers excrete metabolites of various polycyclic aromatic hydrocarbons (PAHs) in bile, and that glutathione transferase (GST)-mediated conjugation is a major detoxification pathway for the PAH benzo[alpha]pyrene. To determine whether hepatocarcinogenesis in these wild fish is associated with induced GST-dependent resistance to carcinogens, we examined the expression of immunoreactive GSTs in liver neoplasms and putatively preneoplastic altered hepatocellular foci from white suckers collected from several polluted sites in western Lake Ontario. Histological sections of liver with altered hepatocellular foci, hepatocellular adenomas, hepatocellular carcinomas, bile duct adenomas and bile duct carcinomas were examined for GST immunoreactivity by the peroxidase-antiperoxidase (PAP) technique with polyclonal antiserum specific for all major GST isoenzyme subunits found in normal liver of white suckers. All bile duct adenomas, bile duct carcinomas and hepatocellular carcinomas were markedly or completely deficient in immunoreactive GST in comparison with surrounding normal hepatocytes. The majority of the hepatocellular adenomas were also deficient. Most altered hepatocellular foci had normal GST staining, but several GST-deficient altered hepatocellular foci were observed. However, none of the preneoplastic or advanced liver neoplasms expressed induced GST, suggesting that carcinogenesis is not associated with selection for GST-dependent resistance. Loss of hepatocellular GSTs may be incidental to neoplastic progression in these fish, or might be important in increasing susceptibility of some preneoplastic populations of hepatocytes to further DNA damage by environmental or endogenous chemicals that are normally detoxified by GSTs.     

胃癌及其转移灶中S100A6基因的表达

目的 分析S100A6在胃癌发展过程中的作用,研究配对胃癌组织以及转移灶中S100A6基因mRNA和蛋白的表达.方法 应用实时定量逆转录聚合酶链反应(QRT-PCR)检测20例配对胃癌组织中S100A6 mRNA的表达,构建208例胃癌患者配对胃癌组织及转移淋巴结病灶的组织芯片,采用免疫组化方法检测S100A6蛋白的表达,并分析其与临床病理因素以及预后之间的相关性.结果 QRT-PCR检测结果显示,20例配对胃癌中,有14例的S100A6 mRNA转录水平升高,平均升高倍数为2.25倍.免疫组化检测结果显示,S100A6在正常胃黏膜中的表达阳性率为34.3%,在癌灶中表达阳性率为84.1%,在淋巴结转移灶中表达阳性率为90.9%,癌灶和淋巴结转移灶表达高于正常胃黏膜(P<0.05).65.5%的胃癌组织中S100A6表达较对应正常胃黏膜升高.癌灶中S100A6表达与肿瘤大小和侵袭程度相关,肿瘤越大表达越高(P=0.022),侵袭越深表达越高(P=0.000),未发现S100A6表达与其他临床病理因素之间的相关性.S100A6表达与预后无关,但相对于正常胃黏膜,癌灶中S100A6表达升高者的预后比表达下降者差.结论 S100A6表达上调是胃癌早期事件,与胃癌的发生、发展相关.     

Epithelial and stromal syndecan-1 expression as predictor of outcome in patients with gastric cancer

The prognostic value of the immunohistochemical expression of epithelial and stromal syndecan-1 was evaluated in 296 patients with gastric carcinoma. Formalin-fixed, paraffin-embedded specimens of gastric adenocarcinomas were stained with mouse monoclonal antibody B-B4 against human syndecan-1. Loss of immunoreactivity (syndecan-1 immunoreactivity correlated with a higher stage of disease (stages II-IV), tumour location in the upper third of the stomach, nodal metastases (N1 or N2), positive stromal syndecan-1 staining, deep tumour penetration (to subserosa or deeper = T2-T4), larger tumour size (> or = 5 cm) and intestinal type of cancer. No correlation between epithelial syndecan-1 immunoreactivity and age, gender, distant metastases, grade of differentiation or Borrmann classification was observed. Positive stromal syndecan-1 immunoreactivity correlated with decreased epithelial syndecan-1 expression, intestinal type of cancer and Borrmann type I. Patients with low epithelial syndecan-1 expression in cancer cells had worse overall survival than patients with strong epithelial syndecan-1 staining (p = 0.0012). Stromal syndecan-1-positive patients had a worse outcome than patients with syndecan-1-negative stroma (p = 0.0193). In Cox multivariate analysis, stromal syndecan-1 immunoreactivity was a prognostic factor independent of TNM stage, surgery for cure and tumour size. Thus, the immunohistochemical expression of syndecan-1 might be a predictor of outcome in patients with gastric adenocarcinoma.     

Correlation of vascular endothelial growth factor expression with fibroblast growth factor-8 expression and clinico-pathologic parameters in human prostate cancer

Vascular endothelial growth factor (VEGF) mediates neo-angiogenesis during tumour progression and is known to cooperate with the fibroblast growth factor (FGF) system to facilitate angiogenesis in a synergistic manner. In view of this, we have investigated VEGF expression in 67 cases of prostate cancer previously characterized for fibroblast growth factor-8 (FGF-8) expression. Cytoplasmic VEGF staining was detected in malignant cells in 45 out of 67 cases. Cytoplasmic staining was found in adjacent stromal cells in 32 cases, being particularly strong around nests of invasive tumour. Positive VEGF immunoreactivity in benign glands was restricted to basal epithelium. A significant association was observed between tumour VEGF and FGF-8 expression (P = 0.004). We identified increased VEGF immunoreactivity in both malignant epithelium and adjacent stroma and both were found to be significantly associated with high tumour stage (P = 0.0047 and P = 0.0002, respectively). VEGF expression also correlated with increased serum PSA levels (P = 0.01). Among positively stained tumours, VEGF expression showed a significant association with Gleason score (P = 0.04). Cases showing positive VEGF immunoreactivity in the stroma had a significantly reduced survival rate compared to those with negative staining (P = 0.037). Cases with tumours expressing both FGF-8 in the malignant epithelium and VEGF in the adjacent stroma had a significantly worse survival rate than those with tumours negative for both, or only expressing one of the two growth factors (P = 0.029). Cox multivariate regression analysis of survival demonstrated that stromal VEGF and tumour stage were the most significant independent predictors of survival. In conclusion, we report for the first time a correlation of both tumour and stromal VEGF expression in prostate cancer with clinical parameters as well as its correlation to FGF-8 expression.