Expression of Inositol, 1,4,5-trisphosphate Receptor in the Nucleus of Human Benign and Malignant Squamous Cells and Its Significance

三磷酸肌醇（Inositol 1，4，5-trisphosphate，IP_3）是细胞内信号转导的重要信使之一，与其受体（IP_3-R）共同调控细胞内钙离子的流动。Ip_3-R蛋白及其编码基因已被Mikoshiba等发现，并制备了与小鼠IP_3-R蛋白反应的单克隆抗体。利用这些单克隆抗体（10A 6、4C 11、18 A10）与人的交叉反应性，我们观察了IP_3-R在人组织和细胞中的分布，特别是在正常皮肤，包括良性鳞状上皮、鳞状细胞癌组织和培养的鳞状细胞癌细胞系中的分布。在本研究项目中，我们检测了10A 6单克隆抗体与细胞核中抗原的特异反应性。培养的鳞状细胞癌细胞的细胞核中最清楚地显示抗原的存在。共聚焦激光扫描显微镜观察显示其在细胞核内呈细颗粒状分布，而在核仁内无分布。通过免疫染色的荧光强度评估的抗原量在细胞系之间存在差异，但与鳞状细胞谱系的分化阶段或发生癌的组织没有相关性。由于各种固定剂的抗原性较弱，未能显示超微结构定位。由于功能分析表明IP_3-R只存在于核膜而不在核内，因此10A 6单克隆抗体可能与IP_3-R蛋白本身以外的交叉反应抗原发生反应。目前正在进一步研究以揭示人类细胞核中抗原的性质。

Inositol 1,4,5-trisphosphate (IP_3) is one of the important messengers of the intracellular signal transduction, and controls the cytoplasmic flow of calcium ion in conjunction with its receptor (IP_3-R). The Ip_3-R protein and its coding gene had been revealed by Mikoshiba et al., and monoclonal antibodies reactive with mouse IP_3-R protein have been produced as well. Using cross-reactivity of these monoclonal antibodies (10A6,4C11,18A10) with the human counterpart we have shown the distribution of IP_3-R in human tissues and cells, especially in normal skin including benign squamous epithel, squamous cell carcinoma-tissues and cultured cell lines of squamous cell carcinoma. In the present researchproject we examined peculiar reactivity of 10A6 monoclonal antibody with an antigen in the nucleus. Presence of the antigen was most clearly shown in the nuclei of cultured squamous carcinoma cells. Observation by confocal laser scanning microscopy revealed its fine granular distribution in the nucleus but not in the nucleolus. The amount of the antigen as assessed by fluorescence intensity of immunostaining differed among cell lines, but there were no correlation with the stage of differentiation in squamous cell lineage or to the tissues where carcinoma arose. Attempt to reveal ultrastructural localization did not succeed because of the weaknees of the antigenicity againt various fixatives. Since funcitonal analysis indicates the presence of IP_3-R only in the nuclear membrane but not in the nucleus, 10A6 monoclonal antibody might react with crossreacting antigen other than IP_3-R protein itself. Further investigation to reveal nature of the antigen in the nucleus of human cells is now in progress.