IN VIVO STUDIES OF A PROLACTIN ANTAGONIST IN SCID MOUSE

描述：（申请人的描述） 人乳腺癌是主要的恶性肿瘤，也是导致乳腺癌的主要原因。 西方社会女性的癌症死亡率。 最近， 人催乳素（hPRL）活性与乳腺癌之间的关系再次受到重视。 在 我们最近的研究，我们已经成功地开发了hPRL受体特异性 通过hPRL分子内的单个氨基酸取代突变的拮抗剂 在位置129处从Gly至Arg（hPRL-G129R）。使用基于细胞的分析，我们 已经证明hPRL-G129 R能够与hPRL受体结合并阻断hPRL受体。 hPRL信号转导。 更重要的是，我们已经证明hPRL-GI29 R是 能够通过诱导 凋亡 在本研究中，我们尝试生产和纯化hPRL-GI 29 R，沿着 与hPRL（作为对照），并将它们用于体内研究。 有两 这一提案的具体目标。 具体目标（1）是生产和纯化 通过以下方法获得大于70mg纯度大于95%的hPRL和hPRL-GI29R， FPLC。在产生hPRL和hPRL-GI29 R后，将在体外对其进行检测， 通过（a）受体结合测定;（B）STAT 5（an 用于PRL的细胞内信号传导分子）磷酸化测定;和（c） 细胞凋亡测定以确保其质量。 具体目标（2）： 使用携带人乳腺癌的SCID小鼠作为hPRL-G129 R的体内研究 模型系统 我们选择使用两种人乳腺癌细胞（T-47D和MCF-7）， 7;来自ATCC），并且将对每种细胞系的两种肿瘤接种模式进行免疫接种。 用于本研究（s.c.监测肿瘤发展，i.p.监测肿瘤生长）。 存活率），以确保体内实验结果。 两天后 肿瘤细胞注射后，将以两个剂量腹膜内注射纯化的hPRL或hPRL-GI29R。 剂量组（1 μ g/g体重和4 μ g/g体重）每日一次，持续两周。 肿瘤发展后，s.c.注射将被解剖 并称重，同时对腹膜内注射肿瘤细胞的小鼠进行跟踪， 生存我们希望hPRL拮抗剂能改善妊娠结局， 人类乳腺癌治疗在不久的将来。

DESCRIPTION: (Applicant's Description) Human breast cancer is the predominant malignancy and the leading cause of cancer death in women from Western society. Recently, the association between human prolactin (hPRL) activity and breast cancer has been re-emphasized. In our recent studies, we have successfully developed a hPRL receptor specific antagonist by a single amino acid substitution mutation within hPRL molecule at the position 129 from Gly to Arg (hPRL-G129R). Using cell-based assays, we have demonstrated that hPRL-G129R was able to bind to hPRL receptor and block hPRL signal transduction. More importantly, we have shown that hPRL-GI29R was able to inhibit breast cancer cell proliferation through induction of apoptosis. In this study, we attempt to produce and purify hPRL-GI29R, along with hPRL (as control), and use them for in vivo studies. There are two specific aims of this proposal. Specific aim (1) is to produce and purify greater than 70mg of hPRL and hPRL-GI29R at greater than 95 percent purity by FPLC. After hPRL and hPRL-GI29R are produced, they will be tested in vitro for their biological activities by (a) receptor binding assay; (b) STAT 5 (an intracellular signaling molecule for PRL) phosphorylation assay; and (c) apoptosis assay to ensure their quality. Specific aim (2) is to carry out in vivo studies of hPRL-G129R using SCID mice bearing human breast cancer as model system. We choose to use two human breast cancer cells (T-47D and MCF- 7; from ATCC) and two modes of tumor inoculation for each cell lines will be used for this study (s.c to monitor the tumor development and i.p. monitor the survival rate) to ensure the in vivo experimental results. Two days after tumor cell injection, purified hPRL or hPRL-GI29R will be injected i.p. at two dose groups (1ug/g body weight and 4ug/g body weight) daily for two weeks. Following tumor development, the tumors after s.c. injection will be dissected and weighed while mice with i.p. injection of tumor cells will be followed for survival. We hope that hPRL antagonist could be used to improve the outcome of human breast cancer therapy in the near future.