Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene.

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer-related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference-mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1-GFP in a GFP expression-dependent manner were selected from established hybridoma clones. Novel anti-CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno-histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti-CAT1 mAbs exhibited internalization activity, antibody-dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW-C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE-1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE-1, and investigated the roles of LYVE-1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)-fused mouse LYVE-1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP-fused mouse LYVE-1 proteins in a GFP expression-dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE-1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA-MB-231 xenograft models. This shows that LYVE-1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

Transcriptional repression of the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1 by a tetR-like gene.

Ralstonia pickettii T1 secretes a poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZ) and a 3-hydroxybutyrate (3HB)-oligomer hydrolase, and extracellularly degrades PHB to produce 3HB. However, it is not clear how the expression of phaZ is regulated. In this study, the mechanism by which phaZ expression is controlled in R. pickettii T1 was examined using a mutant made by the random insertion of a transposon, Tn5. The mutant produced a larger amount of PhaZ than the wild type in nutrient broth or a minimal salt (SM) medium supplemented with succinate. However, there was essentially no difference in the activity or amount of PhaZ in the culture supernatant between the wild type and mutant when the two were grown on 3HB. The gene disrupted by the insertion of Tn5 (epdR) was cloned and its nucleotide sequence was determined. In a BLAST search, epdR showed a high degree of similarity to genes for TetR transcriptional regulators of several bacteria. The introduction of epdR into the wild type and mutant grown on the three media described above decreased the amount of PhaZ. These results indicated EpdR to be involved in the repression of phaZ in R. pickettii T1. A quantitative RT-PCR analysis indicated that mRNA levels corresponded with the activity detected and the amounts of PhaZ in the wild type and mutant. Furthermore, the amount of epdR transcript was inversely proportional to the amount of phaZ transcript. In addition, the existence of a positive element acting on phaZ expression was suggested, because in the mutant lacking EpdR, the amount of phaZ transcript varied in cells grown in SM-3HB, SM-succinate or nutrient broth. Based on the above results, a model for the regulation of PhaZ expression in R. pickettii T1 is proposed.