| Research Abstract |
We have previously reported that normal human salivary gland-derived epithelial cells exclusively express keratinocyte growth factor receptor (KGFR). In the process of malignant transformation of human salivary gland tumors KGFR gene expression disappeared concomitant with the de novo expression of the fibroblast growth factor receptor 1 (FGFR 1) and FGFR4 genes.
In the present study, we introduced wild-type KGFR cDNA or chimeric KGFR/FGFR 1 cDNA, which encoded the extracellular domain of KGFR and the intracellular domain of FGFR 1, into the human salivary adenocarcinoma cell line HSY.Following results were obtained.
1. The growth of HSY_<R2-IIIb> in serum-free medium was significantly decreased compare to that of HSYzeo. FGF-2 exhibited no growth stimulation on HSY_<R2-IIIb> although FGF-1 stimulated the growth slightly. In addition, growth of HSY_<R2-IIIb> was stimulated by KGF for initial 3 days of culture, but then inhibited.
2. TUNEL positive cells, DNA ladder formation and increase
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of CPP32/Caspase-3 activity were observed in HSY_<R2-IIIb> upon stimulation with KGF.
3. FGF-1 and FGF-2 stimulated the phosphorylation of both MEK1/2 and p38 MAPK in HSY and HSYzeo. In contrast, FGF-1, FGF-2 and KGF stimulated MEK1/2 phosphorylation but not P38 and JNK/SAPK in HSY_<R2-IIIb>.
4. Growth of HSY_<R2-IIIb> tumors in athymic mice were dramatically decreased compare to that of HSYzeo. Some clones of HSY_<R2-IIIb> lost their tumorigenicity. By histological examination, HSY_<R2-IIIb> tumors exhibited differentiated morphology such as acinar-like and duct-like structure.
5. KGFR gene therapy starting at one week after tumor inoculation completely cured HSY tumors and that starting at 2-4 weeks significantly inhibited the growth although the tumors did not disappear. Forty eight hours after gene therapy, about 70 % of turmor cells exhibited KGFR expression in nucleus and cytoplasm, and at 4th week from gene therapy, about 20 % of tumor cells still express KGFR protein in cytoplasm and cell membrane.
6. The KGFR tyrosine kinase suppressed the activity of FGF Receptor Substrate 2 (FRS2) and inhibited the growth of HSY by inducing differentiation and apoptosis in vitro and in vivo.
Our results provided a novel and significant insight into the mechanism of KGFR tumor-suppression, and suggest that KGFR gene therapy might be a viable alternative to inhibiting human salivary adenocarcinoma growth. Less
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