Gene Diagnosis and Therapy of Salivary Gland Tumors Targetted for FGFR genes

我们以前曾报道，正常人唾液腺来源的上皮细胞只表达角质细胞生长因子受体（KGFR）。在人涎腺肿瘤的恶性转化过程中，KGFR基因的表达消失，而成纤维细胞生长因子受体1（fibroblastgrowthfactor receptor 1，FGFR 1）和FGFR 4基因的表达则随之消失，本研究引入了野生型KGFR cDNA或嵌合型KGFR/FGFR 1 cDNA，它们分别编码KGFR的胞外区和FGFR 1的胞内区，本研究通过转染人涎腺腺癌细胞株HSY，获得了以下结果. HSY_<R2-IIIb>在无血清培养基中的生长速度明显低于HSYzeo。FGF-1对HSY_2细胞的生长有一定的促进作用，而FGF-2对HSY_2细胞的生长无明显的促进作用<R2-IIIb>。另外，<R2-IIIb>KGF在培养的前3天对HSY_1细胞的生长有促进作用，但随后抑制其生长. TUNEL阳性细胞，DNA梯状条带形成和增加 ...更多信息 结果显示，在KGF刺激下，CPP 32/Caspase-3的活性<R2-IIIb>明显降低. FGF-1和FGF-2刺激HSY和HSYzeo中MEK 1/2和p38 MAPK的磷酸化。相比之下，FGF-1、FGF-2和KGF刺激HSY_4中的MEK 1/2磷酸化，但不刺激P38和JNK/SAPK<R2-IIIb>。与<R2-IIIb>HSYzeo相比，HSY_2肿瘤在无胸腺小鼠中的生长显著降低。部分克隆<R2-IIIb>丧失致瘤性。组织学检查显示，HSY_2<R2-IIIb>肿瘤呈腺泡样、导管样等分化形态.肿瘤接种后1周开始的KGFR基因治疗完全治愈了HSY肿瘤，2-4周开始的KGFR基因治疗显著抑制了肿瘤的生长，但肿瘤没有消失。基因治疗后48 h，约70%的肿瘤细胞在胞核和胞浆中表达KGFR，基因治疗后4周，约20%的肿瘤细胞仍在胞浆和细胞膜中表达KGFR蛋白. KGFR酪氨酸激酶通过抑制FGF受体底物2（FRS 2）的活性，诱导HSY细胞分化和凋亡，抑制HSY细胞的生长，为KGFR抑瘤机制的研究提供了新的思路，并提示KGFR基因治疗可能是抑制人涎腺腺癌生长的一种可行的替代方法。少

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 … More 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