Expression of cellular oncogenes in human salivary gland adenocarcinoma cell line

EGF对细胞生长和EGF受体的影响：在HSG-AZA 3细胞中检测EGF对细胞生长和EGF受体的影响。当细胞经EGF处理后，DNA合成比未经EGF处理的细胞高约3倍。使用抗人EGF受体抗体进行Western blotting，发现约170 kDa的条带为EGF受体，且与未加EGF的条带相比，EGF在170 kDa的条带密度更高。利用EGF受体特异性引物RT-PCR分析EGF受体mRNA。在未受EGF刺激的细胞中观察到142 bp的EGF受体cDNA，该142 bp条带在EGF刺激后12小时达到最大增强。应用特异性磷酸酪氨酸抗体，采用Western blotting分析EGF对EGF受体相关酪氨酸激酶和MAP激酶活性的影响。在未受EGF刺激的细胞中，EGF受体对应区域没有磷酸化条带，但在EGF刺激后5min，在170 kDa附近出现了酪氨酸磷酸化条带。此外，使用酪氨酸激酶特异性底物处理EGF后，EGF受体相关酪氨酸激酶的活性增强。另一方面，MAP激酶在EGF刺激后磷酸化，可通过特异性抗体检测到。此外，EGF刺激15 min后MAP激酶活性升高。c-fos和c-myc原癌基因分析：细胞癌基因c-myc、c-jun或c-fos均可能作为核转录因子发挥作用。通过Western blotting，在62kda附近发现了似乎是c-fos的条带。该波段的强度在EGF处理3 ~ 6小时内增加，然后逐渐降低。RT-PCR检测EGF对c-fos mRNA和c-myc mRNA的影响。EGF对c-fos mRNA和c-myc mRNA的增强作用分别在30分钟和2小时达到最大。

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.