Motomu Kuroki

Homotypic and heterotypic Ca++-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface

Homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, biliary glycoprotein a (BGPa), have been examined. CHO cells transfected with the cDNA for BGPa, CEA, non-specific cross-reacting antigen (NCA) and CGM6 have been used. The BGPa producers showed both homotypic and heterotypic adhesion to CEA and NCA producers. However, they hardly adhered to CGM6 producers. Calcium ion was not required for BGPa-mediated homotypic and heterotypic cell adhesion as well as for the adhesions of other members of CEA family. The results strongly suggested that BGPa may play some important roles through Ca(++)-independent cell adhesion activities.

Sonodynamic therapy of cancer using a novel porphyrin derivative, DCPH-P-Na(I), which is devoid of photosensitivity.

To improve the efficacy of sonodynamic therapy of cancer using photosensitizers, we developed a novel porphyrin derivative designated DCPH‐P‐Na(I) and investigated its photochemical characteristics and sonotoxicity on tumor cells. DCPH‐P‐Na(I) exhibited a minimum fluorescent emission by excitation with light, compared with a strong emission from ATX‐70, which is known to reveal both photo‐ and sonotoxicity. According to this observation, when human tumor cells were exposed to light in the presence of DCPH‐P‐Na(I) in vitro, the least phototoxicity was observed, in contrast to the strong phototoxicity of ATX‐70. However, DCPH‐P‐Na(I) exhibited a potent sonotoxicity on tumor cells by irradiation with ultrasound in vitro. This sonotoxicity was reduced by the addition of L‐histidine, but not D‐mannitol, thus suggesting that singlet oxygen may be responsible for the sonotoxicity of DCPH‐P‐Na(I). DCPH‐P‐Na(I) demonstrated significant sonotoxicity against a variety of cancer cell lines derived from different tissues. In addition, in a mouse xenograft model, a potent growth inhibition of the tumor was observed using sonication after the administration of DCPH‐P‐Na(I) to the mouse. These results suggest that sonodynamic therapy with DCPH‐P‐Na(I) may therefore be a useful clinical treatment for cancers located deep in the human body without inducing skin sensitivity, which tends to be a major side‐effect of photosensitizers. (Cancer Sci 2007; 98: 916–920)

Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family

To clarify the molecular structures of the nonspecific cross-reacting antigens (NCAs) produced by human granulocytes, we cloned cDNAs from libraries of normal white blood cells. A clone, NCA-W272, was found to code a protein similar to NCA of tumor cells. The protein consisted of a signal peptide (34 aa), domain-N (108 aa), -A1 (92 aa), -B1 (86 aa) and -M (29 aa). Similarity of the amino acid sequence of each domain to that of the tumor NCA was 72, 92, 76 and 79%, respectively. COS-1 cells transfected with an expression vector carrying the cDNA synthesized a 70 kDa glycoprotein, which was reactive with anti-NCA antibody and released from cell surface by phosphatidylinositol-specific phospholipase C. Thus the clone NCA-W272 was indicated to encode a new species of NCA distinct from the tumor NCA.

Active production and membrane anchoring of carcinoembryonic antigen observed in normal colon mucosa

Normal colon mucosa was found to produce carcinoembryonic antigen (CEA) quite actively as cancerous tissues do when maintained in an organ culture, although the fresh normal mucosae contained a very small quantity of CEA unlike cancerous tissues. This is consistent with an active expression of CEA mRNA in normal mucosa comparable to that in cancerous tissues actively producing CEA, and suggests that the normal cell product was rapidly released into the lumen of digestive tract and turned into normal fecal antigens (NFAs) previously found in feces. 3H-Labeled precursors of glycophospholipid such as ethanolamine and stearic acid were incorporated into CEA produced by both normal and cancerous tissues, suggesting that CEA in normal mucosa is anchored to the cell membrane through a glycophospholipid as in cancerous tissues.

Expression of four CEA family antigens (CEA, NCA, BGP and CGM2) in normal and cancerous gastric epithelial cells: Up‐regulation of BGP and CGM2 in carcinomas

Four human carcinoembryonic antigen (CEA) family members, CEA (CD66e), non‐specific cross‐reacting antigen (NCA, CD66c), biliary glycoprotein (BGP, CD66a) and CEA gene‐family member 2 (CGM2), are expressed in normal mucosal epithelia of the colon. Expression of BGP and CGM2 has recently been demonstrated to be down‐regulated in colorectal adenocarcinomas. We have now investigated the expression of the 4 CEA family antigens in gastric adenocarcinoma and carcinoma cell lines in comparison with adjacent normal gastric mucosa. The transcripts of the CEA, NCA and BGP genes evaluated by reverse transcription‐polymerase chain reaction were detectable at various levels in all the gastric adenocarcinoma cell lines tested, while CGM2 mRNA was detectable in the cell lines of poorly differentiated but not of well‐differentiated carcinomas. The levels of CEA mRNA in normal gastric mucosa were variable but mostly increased in adenocarcinomas. The sparse expression of NCA observed in the normal tissues was markedly up‐regulated in the carcinomas. In contrast to previous findings on normal and cancerous colonic tissues, the transcripts of CGM2 were totally undetectable and those of BGP were recognized only marginally, if at all, in normal gastric mucosa, while both messages were detected at significant levels in most of the gastric adenocarcinomas. This was confirmed by in situ hybridization. Our findings indicate that expression of the CEA family antigens, particularly that of BGP and CGM2, is differently regulated in epithelial cells of the colon and the stomach. Int. J. Cancer 76:148–153, 1998.© 1998 Wiley‐Liss, Inc.

Tumor growth inhibition by sonodynamic therapy using a novel sonosensitizer

Sonodynamic therapy (SDT) with low-intensity ultrasound combined with a sonosensitizer may be a promising approach to cancer therapy. Use of ultrasound has the advantage of being noninvasive, with deep-penetration properties, and convenient because of the low or no sensitivity of sonosensitizers to light. In this study, SDT with a novel sonosensitizer (a porphyrin derivative) was evaluated in vitro and in vivo. Ultrasound irradiation with a sonosensitizer elicited potent sonotoxicity in vitro without the danger of phototoxicity. The sonotoxic effect was mediated by reactive oxygen species (ROS) and was reduced by ROS scavengers. Cell membrane lipid peroxidation increased significantly just after ultrasound irradiation with a sonosensitizer, but there was no increase in apoptosis. In an in vivo mouse xenograft model, SDT with a sonosensitizer markedly inhibited tumor cell growth. The skin hypersensitivity after light exposure was not observed in a sonosensitizer-treatment group, consistent with the in vitro findings. These results suggest that ROS generated by SDT with a sensitizer can damage tumor cells, resulting in necrosis and prevention of tumor growth. This noninvasive treatment with no adverse effects such as skin sensitivity is therefore promising for therapy of cancers located deep within patients.

Generation, immunologic characterization and antitumor effects of human monoclonal antibodies for carcinoembryonic antigen

We generated fully human mAbs (HmAbs) to carcinoembryonic antigen (CEA) using the KM mouse, which carries a human chromosome 14 fragment containing the entire Ig H chain loci and human κ L chain segments in the mouse genome. Forty‐six hybridoma clones producing HmAbs to CEA were thus obtained by fusing the P3‐U1 mouse myeloma cells with splenocytes of the KM mice immunized with CEA. Among them, 22 clones produced HmAbs that reacted with CEA but not with 3 other CEA‐related cell adhesion molecule (CEACAM) family members, CEACAM1, CEACAM6 and CEACAM8. In 12 HmAbs examined, 8 were IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. The affinity constants for CEA of these HmAbs were comparable to those of the previously prepared mouse anti‐CEA mAbs (MmAbs). BIAcore analyses revealed that 1 and 2 of the 22 HmAbs react with 2 epitopes defined by MmAbs on the domain N and the domain A1 or B1 of CEA, respectively. In the presence of human complement in vitro, 2 HmAbs tested showed substantial cytotoxicity, namely, 50–65%, against CEA‐expressing tumor cells. With human lymphokine‐activated killer cells in vitro, 3 HmAbs tested exhibited 40–65% Ab‐dependent cell‐mediated cytotoxicity against the tumor cells. Moreover, one of the HmAbs induced a significant inhibition of tumor growth when administered to mice xenografted with the CEA‐expressing cells. Considering their lack of immunogenicity to humans, these CEA‐specific HmAbs may be useful for immunotherapeutic approaches as well as for immunodiagnosis.

Carcinoembryonic Antigen–Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors

Purpose: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. Experimental Design: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti–carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. Results:In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC50 against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). Conclusions: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.

Preparation of recombinant MK-1/Ep-CAM and establishment of an ELISA system for determining soluble MK-1/Ep-CAM levels in sera of cancer patients.

The MK-1 antigen, also termed as Ep-CAM, is a membrane glycoprotein that is overexpressed on the majority of tumor cells of epithelial origin and thereby can be used as a target of immunodetection and immunotherapy of cancer. It has previously been shown that several type-I transmembrane proteins, including E-cadherin, ErbB-2 and intercellular adhesion molecule-1 (ICAM-1), may be useful as tumor markers because they are released into the circulation of many cancer patients. To address the question of whether MK-1, the same type-I membrane protein, is also released into the sera, we developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) system by preparing a recombinant MK-1 protein and two anti-MK-1 monoclonal antibodies with different epitope specificities. Using this ELISA, we found that the MK-1 levels in serum samples from healthy volunteers were all less than 2 ng/ml, whereas the Mk-1 levels in sera of about 10% of patients with malignant tumors of various tissue origins were increased to 2-78 ng/ml, indicating that MK-1 is released from tumor cells into the circulation under certain conditions. These findings should be borne in mind when trying to perform passive antibody therapy for cancer using anti-MK-1 antibody.

Identification and comparison of residues critical for cell-adhesion activities of two neutrophil CD66 antigens, CEACAM6 and CEACAM8.

CEACAM6 (CD66c) and CEACAM8 (CD66b) are cell‐adhesion proteins onneutrophils that belong to the human carcinoembryonic antigen (CEA)family. CEACAM6 reveals homophilic adhesion and heterophilic adhesionto other CEACAM family antigens including CEACAM8, CEACAM1, and CEA, whereas CEACAM8 exhibits only heterophilic adhesion to CEACAM6. Here, we investigated and compared structural requirements for the homophilicadhesion of CEACAM6 and heterophilic adhesion between CEACAM6 andCEACAM8 at the amino acid level by using CHO transfectants expressingtheir mutant and chimeric proteins. The NH2‐terminal domain(N‐domain) of CEACAM6 expressed on a CHO cell was suggested to bind the N‐domain of CEACAM6 or CEACAM8 on the opposing cell. Byhomologue‐scanning mutagenesis, we found that the locations of thesequences critical for the adhesion of CEACAM6 to itself and to CEACAM8are overlapped and that they are highly similar but not identical tothe locations of the residues previously shown to be essential for thebinding of CEACAM antigens to Opa proteins of pathogenicNeisseriae. Our findings imply that subtle differences inthe N‐domain sequences determine the specificity of the CEACAM antigenson neutrophils for interaction with the same or different CEACAMantigens and the bacterial proteins.