Immunohistochemical and molecular biological study on expression of diacylglycrol kinase in human origined malignant tumor cells from head and neck

甘油二酯(DG)是重要的第二信使之一，是蛋白激酶C(PKC)的激活剂。DG激酶(DGK)使DG磷酸化，生成磷脂酸，因此DGK被认为是通过减弱DG而调节PKC活性的。最近的研究揭示了哺乳动物物种中几种DGK同工酶的分子结构，并表明大多数同工酶在脑中以不同数量表达。从山形大学医学院解剖与细胞生物学教研室大鼠脑文库(DGKα，-β，-γ，-ζ，-ι)中克隆到5个DGK同工酶基因。用原位杂交组织化学方法研究了DGK同工酶在大鼠脑组织中的表达，但在人类恶性肿瘤细胞，特别是上皮细胞中的表达尚未见报道。在本研究中，我们检测了DGK同工酶在人鳞状细胞癌细胞系中的表达。为了研究Dgkζ的定位和功能作用，我们制备了针对大鼠Dgkζ的特异性抗体，并检测了该抗体的特异性。此外，用DGK抑制剂培养人恶性肿瘤细胞，发现DGK抑制剂有抑制细胞生长的作用。提示DGK可能与细胞生长的分子机制有关。

Diacylglycerol (DG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DG kinase (DGK) phosphorylates DG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed ih the brain in various amounts. Five DGK isozyme cDNA have been cloned from rat brain library (DGKα, -β, -γ, -ζ, -ι) in Department of Anatomy and cell biology, Yamagata University School of medicine. mRNA expressions of DGK isozymes in rat brain have been studied by in situ hybridization histochemistry, but their mRNA expressions in human malignant tumor cells, especially epithelial cells, have not been examined. In this study, we examined the mRNA expression of DGK isozymes in human squamous cell carcinoma cell lines. To study the localization and functional role of DGKζ, we generated the specific antibody against rat DGKζ and examined the specificity of the antibody. Furthermore, human malignant tumor cell has been cultured with DGK inhibitors and it was revealed that DGK inhibitor might suppress cell growth. It shows that DGK may have some relations to the molecular mechanism of cell growth.