Hong-Keun Chung

Detection of cancer cells in peripheral blood of stomach cancer patients using RT–PCR amplification of tumour-specific mRNAs

RT–PCR amplification of tumour‐specific mRNA has been used for the detection of cancer cells in peripheral blood.

Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood.

Purpose: The reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA has been used for the detection of cancer cells in peripheral blood. More recently, an immunomagnetic isolation and reverse transcription polymerase chain reaction (immunobead RT-PCR) was developed which has reportedly significant advantages over the previous RT-PCR analysis. In our study, we compared these two methods using a model set of peripheral blood containing tumor cells under standardized conditions. Material and methods: In order to compare the false positive rate, normal peripheral blood samples from five volunteers were analyzed by both methods. A model set of peripheral blood containing tumor cells was established by adding SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PCR products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobead RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic beads before the RT-PCR analysis. Result: The immunobead RT-PCR yielded no non-specific band, while the regular RT-PCR using total RNA did show non-specific band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobead RT-PCR allowed the detection of 101 tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 102 tumor cells in 1 ml of peripheral blood. Conclusion: The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our model system.

Expression and Characterization of Anti-NCA-95 scFv (CEA 79 scFv) in a Prokaryotic Expression Vector Modified to Contain a Sfi I and Not I Site

The CEA 79 antibody has been used in bone marrow scintigraphy for the differential diagnosis of skeletal tumors and the evaluation of the bone marrow status of patients with various hematological disorders. The specific localization of radio-labeled CEA 79 antibody in bone marrow depends on its reactivity with NCA-95 (nonspecific cross-reacting antigen-95) present on the surface and in the cytosol of human granulocytes and myelopoietic cells. To make a CEA 79 scFv molecule that would be less immunogenic and more penetrating than the intact mouse immunoglobulin, we constructed a pRSET Sfi I/Not I expression vector. The scFv gene was then excised from a pCANTAB 5 E phage display vector by digestion with Sfi I and Not I and inserted into the pRSET Sfi I/Not I expression vector. Upon transformation of a BL21(DE3)pLysS strain of E. coli, CEA 79 scFv became expressed in inclusion bodies requiring a renaturation process for solubilization. The final yield of CEA 79 scFv was 5 mg per a liter of culture. The refolded CEA 79 scFv exhibited an affinity (Kd = 2.1 x 10(-9) M) equivalent to that of the original CEA 79 antibody (K(d) = 3.3 x 10(-9) M) and the same immunoreactivity to CEA and NCA-95 in Western blots and in immunohistochemical staining experiments.

Development of bone marrow immunoscintigraphy using a Tc-99m labeled anti-NCA-95 monoclonal antibody

We evaluated the monoclonal antibody CEA-79.4 against carcinoembryonic antigen as an immunoscintigraphic agent for assessing the state of the bone marrow. Western blotting of human granulocyte extracts with the antibody could confirm that the binding was with the epitope of NCA-95. Immunocytochemical staining of bone marrow aspirates revealed specific uptake of this antibody by granulopoietic cells. The affinity constant was 2-9 x 10(9) L/mol. Immunoscintigraphy using 99mTc-labeled CEA-79.4 in a normal volunteer revealed high uptake in the bone marrow as compared to other organs.

Concentration and distribution of tumor associated antigens TAG-72 and CEA in stomach cancer

We measured the concentration and distribution of tumor associated antigens, TAG-72 and CEA, in stomach cancer byin vitro quantitative autoradiography (IV-QAR). Frozen sections of 33 specimens were incubated with varying concentrations of125I-labeled CEA-79.1 and B72.3 antibodies specific for carcinoembryonic antigen (CEA) and tumor-associated glycoprotein-72 (TAG-72), respectively. Computer analysis of specific antibody binding gave maximal binding values which were equal to the concentrations of the antigen or epitope. TAG-72 was detected in 25 specimens, at a concentration ranging from 8.4 to 562.9 pmol/g. CEA was detected in 32 of the 33 specimens and its concentration ranged from 8.8 to 525.3 pmol/g. The distribution of TAG-72 by IV-QAR coincided with that of the tumor cells in 41.4% of the pathologic lesions. The distribution of CEA coincided with the tumor cells in 80.5% of pathologic lesions, nearly twice the TAG-72. The concentration of TAG-72 was significantly higher in mucinous adenocarcinoma and mucin containing adenocarcinomas than other types of adenocarcinomas. There was no significant difference in the concentration of CEA among the pathologic types of stomach cancer. In summary, stomach cancer exhibited wide variations in TAG-72 and CEA expression. CEA expression was more frequent and homogeneous than TAG-72.

Generation and Characterization of IgG Monoclonal Antibodies Specific for Malondialdehyde

Numerous studies have indicated that the oxidative modification of low-density lipoprotein (LDL) plays a critical role in the pathogenesis of atherosclerosis. Malondialdehyde-modified LDL (MDA-LDL) is one of the candidate oxidative products. Therefore, to allow the assessment of oxidized LDL in human serum, we developed monoclonal antibodies for MDA-LDL. Two of these-MDA1 and MDA2-bound to oxidized LDL but not to native LDL by Western blot analysis. The murine monoclonal antibodies to oxidized LDL have potential clinical implications, as imaging agents, for defining the compositions of atherosclerotic vessels in vivo.

Production and characterization of a monoclonal antibody specific to the human 70-kDa heat shock protein.

Heat shock protein 70 (hsp 70) plays major roles in apoptosis prevention and thermotolerance as well as molecular chaperoning. It is also expressed on the surface of human tumor cells, but not on normal cells, suggesting that hsp70 may be some tumor-associated antigen. To investigate the diverse functions of the protein species, various types of transgenic mice or cell models overexpressing human hsp70 have been made. In these models a monoclonal antibody (MAb) specific for the human hsp70 is highly desirable to distinguish the human from the endogenous mouse hsp70. It proved difficult to make this species-specific MAb, because the hsp70 homologues are members of a family of highly conserved, abundant, and ubiquitous proteins expressed in organisms ranging from bacteria to humans. In the present study, we prepared four MAbs against human hsp70. Three, HD 5, HD 7 and HD 11, recognize human and mouse hsp70. One, though, HD 8, recognizes human hsp70, but not mouse hsp70. By Western blot analysis of hsp70 deletion mutants, the epitope of the HD 8 MAb was determined as the 585-616 amino acid region of the human hsp70, a region with relatively low homology to mouse hsp70.

A novel tumor-associated mucin of gastrointestinal carcinoma

Abstract Purpose: To identify a new tumor-associated antigen, a monoclonal antibody, SC142, was produced by immunizing mice with a stomach cancer cell line. The tumor specificity of mAb SC142 was studied by immunohistochemical staining, and the biochemical characteristics of this new gastrointestinal tumor-associated antigen were also studied. Methods: The expression of SC142-reactive antigen was investigated in various cancers by immunohistochemical staining. The SC142-reactive antigen was characterized by immunoblotting, sodium metaperiodate treatment assay, O-glycanase digestion assay, and lectin binding assay. Results: The SC142-reactive antigen was highly expressed in 78% of gastric cancers (29/37) and 87% of colon cancers (27/31). No normal colon or stomach tissues remote from the tumor were positive for the antigen. The antibody also reacted with other tumors of epithelial origin such as lung squamous cell cancer (2/4), breast ductal cancer (2/20), bladder transitional cell carcinoma (4/6), and uterine cancer (3/16). Western blot analysis of the antigen revealed glycoprotein(s) which migrated as a smear ranging from the origin of the gel to about the 80 kDa region. The reactivity of this antigen with SC142 was reduced by sodium metaperiodate treatment or O-glycanase digestion, but not by N-glycanase, suggesting that the epitope is an O-glycan. In lectin-binding assay, this antigen reacted only with wheat germ agglutinin but not with Ricinus communis agglutinin, Datura stramonium agglutinin, and Sambucus nigra agglutinin. Conclusions: Our findings indicate that the antigen defined by SC142 is a tumor-associated antigen that could differentiate the gastrointestinal cancer cells from the normal cells. Therefore, SC142 may become a valuable tool for the immunohistochemical diagnosis and tumor immunoscintigraphy of the gastrointestinal cancer patients.

Radioimmunoscintigraphy of advanced gastrointestinal carcinomas employing I-131 labeled CEA-79 monoclonal antibody

CEA-79 is a murine IgG2a type monoclonal antibody (MoAb) generated using purified CEA from culture supernatants of a human colon cancer cell line, LS174T. The association constant and immunoreactivity of the I-131 labeled CEA-79 ranged from 2.0 to 3.2 ×109 l/mole, and from 54 to 74 %, respectively. The purpose of this study was to evaluate the feasibility of radioimmunoscintigraphy employing MoAb CEA-79 in patients with advanced gastrointestinal carcinomas. Two mgs of MoAb CEA-79 was labeled with 111 MBq (3 mCi) of I-131, and infused intravenously in 6 stomach cancer and 16 colon cancer patients. Out of 6 patients with stomach cancer, immunoscintigraphy was able to detect the tumors in 4 cases. However, immunoscintigraphy found out tumors in all patients with colon cancer. Moreover, 1 patient with stomach cancer and 2 patients with colon cancer showed increased uptake of MoAb in the tumor lesions despite normal serum levels of CEA. We could conclude that this antibody has a potential as a new imaging agent for the diagnosis of gastrointestinal carcinoma.

Metabolic loading of guanosine induces chondrocyte apoptosis via the Fas pathway

Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.