Sanetaka Shirahata

Telomere Shortening in Cancer Cells by Electrolyzed-Reduced Water

Electroly-reduced water (ERW) which is produced near cathode during electrolysis of water scavenges reactive oxygen species and protects DNA from oxidative damage (Shirahata et al., 1997). Most of cancer cells exhibit high telomerase activity to elongate telomere length, insuring their immortality. Here we found that cultivation of human lung adenocarciomaA549, human uterine cancer HeLaand human normal fibroblast TIG-1 cells were growth inhibited in medium containing ERW and drastic morphological changes occurred in A549 and HeLa cancer cells but not in TIG-1 cells. Telomerase activity did not change but telomere length became shorter depending upon cell division in medium containing ERW. Telomere binding activities of telomere binding proteins in cancer cells decreased in medium containing ERW, suggesting that ERW inhibit the binding of’ telomerase to telomere region via telomere binding proteins, resulting in the shortening of telomerelength.

Induction of Rice Allergen-Specific IgE Synthesis by KU812 Cells

In vitro IgE class switching could be induced through co-culture of CD40L-expressing KU812 cells and CD40-expressing B cells in the presence of IL-4 or IL-13. It has been generated several B cell lines, which produce rice allergen (RA)-specific IgM antibody by in vitro immunization (IVI) using peripheral blood lymphocyte (PBL). In this study, induction of RA-specific IgE antibody by KU812 cells was attempted. Before co-culture, we determined the CD40 expression in RA-specific B cell lines, RA9G11 and the CD40 ligand (CD40L) expression in activated KU812 cells by treatments with phorbol myristate acetate (PMA) and ionomycin for 6 hrs. Flow cytometric analysis shown that RA9G11 and activated KU812 cells expressed high level of CD40 and CD40L, respectively. RA9G11 cells were cultured with activated KU812 cells for 12 days in the presence of IL-4 for IgE class switching. Mature Ce mRNA level and RA-specific IgE spot forming cells (SFC) were observed in all culture condition, and especially, high level of RA-specific IgE synthesis was determined the same ratio of RA9G11 and activated KU812 cells in the presence of 50U IL-4. Therefore, induction of RA-specific IgE synthesis by activated KU812 cells can be contributed in the application for allergic therapy and prevention.

Acquired Autoantibodies from Human Plasma B Cells by Light Chain Shifting

HTD8 is one of concanavalin A-resistant variant subclones of a human plasma B cell line. We have shown that HTD8 expresses various new light chains replacing the original light chain at a high rate. This process of replacing the original light chain in antibody secreting cells was called “light chain shifting”. The subclones of transformed-HTD8 cells by introducing certain human antibody heavy chain gene, expressed new light chains which led to altered antigen binding in both specificity and affinity. These results raise the possibility that plasma B cell can be altered to be generate autoantibodies. We examined this possibility by assessing an autoantigens binding of hybrid antibody molecules from the transformed-HTD8 cells repertoire. The hybrid antibodies were obtained by panning against dsDNA and further examined with other autoantigens. Some of the antibodies produced from the transformed-HTD8 cells also express a different light chain and shown to be of high affinity with double-stranded DNA (dsDNA), human thyroglobulin, and Fc fragment of human IgG. These results indicated that light chain shifting can be utilized to generated autoantibodies with high affinity and polyspecificity to various autoantigens by an antibody having no significant affinity for autoantigens.

A Retrovirus-Associated Antigen Expressed in Human Lung Carcinoma

A cDNA fragment, detectable in a human lung adenocarcinoma cell line A549, was amplified using PCR technique with oligonucleotide primers specific to cytochrome c from Candida krusei which was recognized by the lung cancer-specific human monoclonal antibody HB4C5. We screened the A549 cDNA library with the PCR product as a probe, and isolated a cDNA clone, named PRI-23. Sequencing analysis of the PRI-23 clonal cDNA revealed a 570 bp open reading frame (ORF) which encodes a 190 amino acid residue near the 3′-terminal region of the cDNA. A partial nucleotide sequence in the ORF exhibited about 90% homology to the long terminal repeat (LTR) sequence of the human endogenous retrovirus HERV-K10. Northern blot analysis indicated that the antigen mRNA was also detected in stomach and mammary carcinoma lines, but was not in normal fibroblast. These results suggest that this antigen may be tumor-associated and is expressed during or after carcinogenesis.