Definition of the expression of the human carcinoembryonic antigen and non-specific cross-reacting antigen in human breast and lung carcinomas

Mouse cell lines transfected with carcinoembryonic antigen (CEA) and with 2 other members of the human CEA gene family, non-specific cross-reacting antigen (NCA) and biliary glycoprotein (BGP), were used to analyze the specificity of several monoclonal antibodies (MAbs). MAbs COL-1 and COL-6 were shown to react with the transfected CEA gene product but not with NCA, confirming previous results. Cells expressing the transfected BGP gene product also failed to react with COL-1 and COL-6. The MAb B6.2 reacted with cells expressing the NCA gene product but not with those expressing CEA or BGP. The MAb B1.1 reacted strongly with the transfected CEA and BGP gene products but only weakly with the NCA gene product. These antibodies were then utilized in the histochemical analysis of a number of primary and secondary breast and lung tumors. The results indicate that a majority of breast and lung tumors express CEA, and nearly all breast and lung tumors express NCA. Fairly homogeneous expression of CEA and NCA was seen in the majority of both breast and lung tumors. Our results indicate that CEA may be an important target for immunotherapy in a large number of patients with breast and lung tumors.     

Shortened Telomere Length in Hepatocellular Carcinomas and Corresponding Background Liver Tissues of Patients Infected with Hepatitis Virus

The telomere length in 20 surgically resected human hepatocellular carcinomas (HCCs) and adjacent non-cancerous, livers with hepatitis virus infection were investigated. All the HCC samples examined demonstrated shorter telomere length than the corresponding non-cancerous liver tissues, the respective average values being 5.4 kbp and 8.8 kbp ( P < 0.001). The shortening of telomere length was most prominent in HCCs larger than 30 mm in diameter, and in both tumors and non-cancerous livers it was more marked with hepatitis B virus as compared with hepatitis C virus infection. These results indicate that telomere shortening is associated with not only progression, but also development of HCC, and there is a possible difference in the nature of the association in patients with hepatitis viruses of B and C types.     

Expression of Lewis blood group antigens in cancerous and non-cancerous liver

The expression of Lewis blood group antigens, Lewisa, Lewisb, Lewisx and Lewisy, in 40 non-cancerous livers and in 20 hepatocellular carcinomas (HCC), 5 cholangiocarcinomas (CC), and 6 combined hepatocellular and cholangiocarcinomas (Comb) was studied by immunohistochemistry using monoclonal antibodies. Although normal hepatocytes did not express any of the four Lewis antigens, Lewisy expression was detected in some hepatocytes at the periphery of pseudolobules in cirrhotic liver. While Lewisx in bile duct epithelial cells was undetectable in normal livers, it was detected in some cases with non-cancerous liver diseases. Lewisx and Lewisy were detected in 30% of HCC and 100% of CC and Comb. Non-cancerous bile duct epithelial cells in almost all instances were stained strongly by anti-Lewisa and/or anti-Lewisb antibody. Proliferating bile ductules within Glisson's sheath and pseudolobules could be clearly detected by Lewisa and Lewisb staining. Lewisa and Lewisb were never detected in non-cancerous hepatocytes, and were only rarely detected in HCC, but were expressed in most cases of CC and Comb. These results suggested that Lewisa and Lewisb are useful markers for differentiation towards biliary epithelial cells in the liver, and that Lewisx and Lewisy expression might be associated with states of increased or altered cell proliferation.     

Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis

The aim of this study was to examine the significance of aberrant DNA methylation, the participation of which in genetic instability is controversial, in hepatocarcinogenesis. The DNA methylation status of the region around the promoter of the E-cadherin tumor suppressor gene, which is located on 16q22.1, and the allelic status at the D16S421 locus, which is adjacent to the E-cadherin locus, were examined using microdissected liver specimens from 38 hepatocellular carcinoma (HCC) patients. Almost all of the non-cancerous liver tissues showed histological findings compatible with chronic hepatitis and cirrhosis, which are considered to be precancerous conditions. DNA hypermethylation was detected in 61% of the non-cancerous liver tissues. The incidence of DNA hypermethylation in the non-cancerous liver tissues of patients with HCCs also showing DNA hypermethylation (72%) was significantly higher than that of patients without DNA hypermethylation in their HCCs (53%, P<0.05). Loss of heterozygosity (LOH) at the D16S421 locus was detected in 35% of the non-cancerous liver tissues. The incidence of LOH in the non-cancerous liver tissues of patients with HCCs also showing LOH was 78%, whereas LOH was not detected in non-cancerous liver tissues of patients without LOH in their HCCs. Fifty-two percent of the non-cancerous liver tissues showed both or neither of DNA hypermethylation and LOH; the incidence of DNA hypermethylation alone in noncancerous liver tissue was 41%. The incidence of LOH alone in non-cancerous liver tissue (7%) was significantly lower compared to those of the former two cases (P<0.0001). These data suggest that aberrant DNA methylation participates in the precancerous stage of hepatocarcinogenesis by preceding, or causing, LOH.     

Carcinoembryonic antigen-like substances of human bile. Isolation and partial characterization.

Three species of carcinoembryonic antigen (CEA)-like macromolecules, called the biliary glycoprotein I, II and III (BGP I, II and III) were identified in human bile. BGP I was found in normal gall-bladder bile and hepatic bile but not in bile subjected to inflammation ("white bile"). It was immunologically related to CEA and NGP (the "normal CEA-like glycoprotein". Synonyms: NCA, CCEA-2, etc.). BGP I differed immunologically from CEA in that it lacked the tumor-associated determinants of CEA. It was different from NGP (and CEA) in that it contained BGP I specific determinants as revealed by anti-BGP I antibodies. In bile from gallbladders with obstructed outlet and subjected to cholecystitis, a non-malignant inflammatory process, BGP I was replaced by BGP II and BGP III. Immunologically and physicochemically, BGP II and BGP III appeared to be closely similar to NGP and CEA, respectively.     

Microsatellite instability correlates with normal expression of cyclin E in hepatocellular carcinomas

It has been reported that microsatellite instability (MSI) strongly correlates with carcinogenesis and cancer progression. In the present study, we studied the incidence of MSI at 5 polymorphic microsatellite markers (D5S406, D13S153, D16S402, D17S796, and poly(A) tract BAT26), the expression of G1 cyclins (cyclin A, cyclin D and cyclin E), and Ki-67 labeling index in 30 surgically resected hepatocellular carcinomas (HCCs) and their adjacent non-cancerous tissues. The results of analysis showed that 43% of HCCs exhibited MSI in one locus, 10% in two loci, and 3% in three loci. Overexpressions of cyclin E and cyclin A were observed in 57% and 83% of HCCs, respectively. MSI in HCCs, however, correlated with normal expressions of cyclin E and cyclin A and with a low labeling index of Ki-67. Thus, patients with HCCs exhibiting MSI at these 5 markers may have less involvement of G1/S disregulation and may have better prognosis than other patients with HCC.     

Determination of the specificities of monoclonal antibodies recognizing members of the CEA family using a panel of transfectants

Carcinoembryonic antigen (CEA), one of the most clinically important tumor markers, is mainly used in the post-surgical surveillance of patients with colorectal carcinomas. CEA belongs to a large protein family, which includes cross-reacting antigens, e.g., non-specific cross-reacting antigens (NCAs) and biliary glycoprotein (BGP) as well as pregnancy-specific glycoproteins (PSGs). The genes encoding these proteins can be subdivided into the CEA and PSG subgroups. The members of the subgroups share antigenic determinants and show high similarity in amino-acid sequences. Their derived secondary structures show them to belong to the Immunoglobulin superfamily. Due to the close relationship of the members of the CEA subgroup, it is very difficult to distinguish between the individual members with MAbs. Here we have used flow cytometric analysis of transfectants expressing individual members of the CEA subgroup as an alternative approach to determine the specificities of 13 MAbs. This allows us to examine the specificities of these antibodies for members of the CEA family, even of those which have not yet been characterized at the protein level. In addition, binding of the MAbs to NCAs expressed by polymorphonuclear cells (PMN) was tested by Western-blot analysis, immunoprecipitation and flow cytometry. Four antibodies bound exclusively to NCA-50/90 and one MAb (80H3) only to NCA-95. MAb 4/3/17 recognizes CEA and BGP on the surface of transfectants and NCA-160 from granulocytes. We assume that NCA-160 is a product of the BGP gene. On granulocytes, which do not express CEA, MAb 4/3/17 is specific for NCA-160 (BGP). Mutual inhibition of the MAbs binding to NCA-50/90 revealed 3 different epitope groups.     

Expression of retinoid X receptor alpha is decreased in 3'-methyl-4-dimethylaminoazobenzene-induced hepatocellular carcinoma in rats

The identification of the specific molecular targets, which underlie liver carcinogenesis is essential for the establishment of an effective strategy for the prevention and/or treatment of hepatocellular carcinomas (HCCs). We previously found that a malfunction of RXRalpha due to its aberrant phosphorylation was associated with the development of HCCs. However, it has remained unclear whether the abnormalities in the expression of RXRalpha or the other retinoid receptors play a role in the early stage of liver carcinogenesis. The present study was designed to determine whether alterations in the expression of RXRalpha and the other retinoid receptors RARalpha and RARbeta are involved in hepatocarcinogenesis using a 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced rat liver carcinogenesis model. We found that immunohistochemical expression of RXRalpha was decreased in liver cell tumors (HCCs and adenoma) and glutathione S-transferase placental form (GST-P)-positive foci, which is a precancerous lesion of HCC, when compared with the non-cancerous tissues. Western blot and RT-PCR analyses revealed a progressive decrease in the expression levels of RXRalpha, RARalpha, and RARbeta proteins and their mRNAs in 3'-MeDAB-induced HCCs and their surrounding tissues, when compared with the normal liver tissues from the control group. Moreover, the expression level of beta-catenin, the heterodimeric partner for both RXRalpha and RARalpha, was immunohistochemically observed in the cytoplasm and, in some cases, in the nucleus of HCC cells. The nuclear expression of cyclin D1, the downstream target molecule of beta-catenin, was also increased in HCC cells when compared with their adjacent normal appearing tissues. Our findings suggest that loss of retinoid receptors, especially RXRalpha, plays a critical role in the chemically-induced rat liver carcinogenesis and this might be associated with the activation of beta-catenin-related signaling pathway.     

乙肝病毒相关的肝癌p16异常甲基化

Objective： To investigate the potential linkage between high rate of p16 methylation and hepatitis B virus （HBV） infection, methylation status of p16, HBV infection markers in serum and HBV-DNA replication level in cancerous and non-cancerous tissue of 32 cases of hepatocellular carcinomas （HCC） with HBV infection and 12 HCCs without HBV infection were examined. Methods： p16 methylation was detected with methylation-specific polymerase Chain reaction （PCR）, and HBV markers were examined with real-time PCR and immunologic method. Results： Methylation of p16 promoter was found in 31 （70.5%） of 44 cancerous tissues of HCC, 2 （16.7%） of 12 HCC without HBV infection, 29 （90.6%） of 32 HCCs with HBV infection marker, p16 methylation was detected in 5 （83.3%） of 6 HCCs positive for HBsAg and HBeAg, 17 （94.4%） of 18 HCCs positive for HBsAg and negative for HBeAg, 7/8 （87.5%） of HCCs positive for other HBV infection markers, such as HBsAB, HBcAb, HBeAb. p16 methylation products were also found in non-cancerous tissues of 4 cases of HCCs with HBV infection, not detected in non-cancerous tissues without HBV infection. HBV-DNA was detected in cancerous tissues of 29/32 （90%） HCCs with HBV infection. Surprisingly, Methylation product of p16 promoter was found in all cases （29/29） of HCCs with detectable HBV-DNA in neoplastic tissue. Conclusion： Persistent HBV infection may promote p16 hypermethylation, suggesting that HBV, via enhancing the aberrant methylation of p16, indirectly involved in development of HCC.     

肝癌DKK1基因的表达及突变分析

目的:探讨中国地区人肝癌发生发展过程中有无DKK1基因突变。方法:Northernblot方法分析12例肝癌患者癌和癌旁肝组织DKK1mRNA表达水平。采用PCRSSCP方法,检测20例肝癌患者(包括癌和癌旁肝组织)及8种人肝癌细胞株中有无DKK1基因突变,并采用DNA测序对PCRSSCP突变分析结果加以验证。结果:Northernblot显示12例肝癌患者中7例存在癌组织DKK1mRNA高表达而癌旁肝组织微弱表达或不表达。突变检测发现20例肝癌患者及8种人肝癌细胞株中仅1例肝癌患者存在DKK1基因的同义突变(单核苷酸多态现象)。结论:DKK1在中国人肝癌中存在高表达,但未发现突变发生,提示DKK1基因参与肝癌的癌变过程可能并不依赖于该基因的突变。     

The Clinicopathologic Significance of BAF250a (ARID1A) Expression in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and lethal human cancers. Recently, exome sequencing has revealed that mutation of ARID1A is frequent in HCC. Herein, we determined the clinicopathologic significance of ARID1A expression in HCC. We detected the level of mRNA and protein expression of ARID1A in 12 paired HCC tumors and adjacent non-cancerous tissues by quantitative real-time PCR and immunohistochemistry (IHC). In addition, we determined the expression of BAF250a on 121 HCC tumors by IHC and assessed the association between BAF250a expression and clinicopathologic and prognostic features. The levels of ARID1A mRNA were significantly elevated in 10 of 12 HCC tumors compared with adjacent non-cancerous tissues. The level of BAF250a protein expression was higher in 10 of 12 HCC tumors compared with adjacent liver tissues. IHC indicated that 12.17 % of HCC tumors (14/115) were BAF250a-negative. Loss of BAF250a was significantly associated with larger tumor size, but not associated with other clinicopathologic features. There was no significant difference in disease-free or overall survival between BAF250a-positive and BAF250a-negative patients. Most HCCs had an increased level of ARID1A mRNA and BAF250a expression. Loss of BAF250a was significantly more frequent in larger HCC tumors, but had no prognostic significance.     

CEA-related proteins on human urothelial cell lines of different transformation grades.

CEA family proteins from human urothelial cell lines of different transformation grades were characterized by flow cytometry and Western blotting using monoclonal antibodies: 26/3/13, D14HD11, 9A6 and 4/3/17. The following observations were made: (i) the urothelial cell lines, representing transformation grade III (TGr III, tumorigenic, invasive cells), were characterized by the presence of a component with molecular mass 110-135 kDa, most probably representing biliary glycoprotein (BGP); (ii) BGP was absent in non-tumorigenic and non-invasive TGr II urothelial cell lines; (iii) a protein band with apparent molecular mass 180 kDa, and migrating as a CEA standard was detected in only one of seven urothelial cell lines analyzed; (iv) a broad band of apparent molecular mass migrating at 65-90 kDa, probably representing NCA-50/90, was found in two tumorigenic and invasive cell lines, HCV 29T and Hu 1703He.     

RhoC基因的过量表达与肝细胞癌的侵袭转移

目的　研究探讨RhoC基因mRNA和蛋白的过量表达与肝细胞癌 (HCC)的发生发展和侵袭转移的关系。方法　采用逆转录聚合酶链反应 (RT PCR)和Western印迹法 ,检测 2 5例HCC及其癌旁肝组织、4例门静脉主干癌栓或肝外转移灶中RhoCmRNA和蛋白的表达 ;同时采用PCR产物单链构象多态性 (PCR SSCP)银染法检测RhoC基因的突变情况。结果　HCC组织和癌旁肝组织中均可检测到RhoC基因mRNA和蛋白的表达 ,RhoCmRNA在HCC组织中的表达较癌旁肝组织高 ,其吸光度(A)比值分别为 1.8± 1.1和 1.0± 0 .7,差异有显著性 (P <0 .0 1) ;RhoC蛋白在HCC组织中的表达同样高于癌旁肝组织 ,其A比值分别为 33992± 10 384和 17342± 9998,差异有显著性 (P <0 .0 1)。 4例门静脉主干癌栓或肝外转移灶中的RhoC基因mRNA和蛋白的表达量分别为 3.3± 0 .5和 6 386 5±9116 ,较HCC肝内病灶高 ,差异有显著性 (P <0 .0 1) ;RhoCmRNA和蛋白在分化较差、结节数较多、有静脉浸润或伴有肝外转移灶的HCC中过量表达 (P <0 .0 5 )。全部HCC经PCR SSCP银染法分析均未发现异常泳动条带。结论　RhoC基因的过量表达与HCC的发生发展和侵袭转移密切相关 ,且可能是通过过量表达而非突变发挥作用。