| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
|
|---|
| Research Abstract |
The effect of EGF on the cell growth and EGF receptor : The effect of EGF on the cell growth and EGF receptor was examined in HSG-AZA 3 cells. When the cells were treated with EGF,DNA synthesis was approximately 3-fold higher than that without EGF treatment. Western blotting using anti-human EGF receptor antibody showed the band around 170 kDa estimated as EGF receptor, and EGF showed the more high density band with 170 kDa as compared to that without EGF.EGF receptor mRNA was analyzed by RT-PCR using specific primers for EGF receptor. EGF receptor cDNA with 142 bp in unstimulated cells was observed and a maximal enhancement of this 142 bp band was obtained 12 hr after EGF stimulation.
The effect of EGF on activities of EGF receptor-associated tyrosine kinase and MAP kinase : Autophosphorylation of EGF receptor was analyzed by Western blotting using a specific antibody against phosphotyrosine. Although there was no phosphorylated band in the region corresponding to EGF receptor in unstimulated cells, a phosphorylated tyrosine band appeared around 170 kDa 5 min after EGF stimulation. Furthermore, the activity of EGF receptor-associated tyrosine kinase was enhanced after EGF treatment using the specific substrate for tyrosine kinase. On the other hand, MAP kinase phosphorylated after EGF stimulation was detected by a specific antibody against phospho-MAP kinase. In addition, the activity of MAP kinase increased 15 min after EGF stimulation.
Analysis of c-fos and c-myc proto-oncogenes : Cellular oncogenes, c-myc, c-jun or c-fos which are induced after simulation of growth factors, may play a role as nuclear transcriptional factors. By Western blotting, the band around 62 kDa which seems to be c-fos was found. The intensity of this band increased during 3-6 hr treatment of EGF and then gradually decreased. The effect of EGF on c-fos mRNA and c-myc mRNA was also examined by RT-PCR.EGF caused maximal enhancement of c-fos mRNA at 30 min and that of c-myc mRNA at 2 hr.
|
