| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
The rat thyroid cancer cell line, FRTC,was used to investigate a novel therapy for human thyroid cancer. Since FRTC cells have TSH receptors (TSH-R), the complex between anti-TSH-R antibodies and anti-tumor agents may be a useful tool to kill FRTC cells. To make antibodies to rat TSH-R,full length rat TSH-R cDNA was inserted into a procaryote expression vector pGX2T (Pharmaia). TSH-R,which should be expressed as a protein fuesd with glutathion S-transferase (GST), formed inclusion bodies in E.coli, and was very hard to dissolve. Amounts of recovered TSH-R-GST fusion protein from a glutathion Sepharose 48 column were too little to immunize. A mammalian expression vector, pEF321-FL,was then employed to express TSH-R cDNA in Cos 7 cells. However, amounts of TSH-R protein recovered from Cos7 cells were also too little for the immunization. Antiserum against synthesied TSH-R peptides were then used. However, their effect was not strictly specfic for FRTC,and it was too expensive to purify s
… More
pecific IgG antibodies against TSH-R from the serum.
Antisense oligonucleotides against rat c-myc mRNA were then tried to inhibit the growth of FRTC cells. The antisense oligonucleotide partially inhibited the proliferation of FRTC cells, but the sense oligonucleotide had no effect. The phosphorothioated antisense oligonucleotide suppressed the proliferation of FRTC cells almost completely. Antisense oligonucleotides, therefore, may be an useful tool to inhibit the growth of tumors which express c-myc proto-oncogene if several problems such as low specificity or fragility to endonucleases are solved.
Inhibitors to protein kinase C (PKC) such as staurosporin or H7 suppressed the growth of FRTC.It is of interest that phorbol 12-myristate 13-acetate (PMA), which stimulates PKC,also inhibited the growth of FRTC.After the PMA stimulation in vitro, the expression of PKCalpha and PKCbeta_2 mRNA as well as TSH-R and IGF-1-R mRNA in FRTC cells decreased. These observations suggest that PKC may play an important role in the growth of FRTC cells.
Cytotoxic T cells to FRTC were induced by the immunization of FRTC cells to Fisher rats. When spleen cells were cultured with FRTC,they showed little cytotoxicity. However, when they were stimulated with culture supernatants of lectin-stimulated spleen cells from immunized Fisher rats, the cytotoxic activity greatly increased. Supernatants themselves also inhibited the proliferation of FRTC.The factor (S) in the supernatants to supperss the growth of FRTC remains to be elucidated. Less
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