MECHANISMS OF TUMOR CELL INVASION

肿瘤细胞通过基底膜和间质侵入 组织允许肿瘤转移并在体内新的部位定植。 然而，调节细胞侵袭的方式完全是不确定的。 未知最近的研究表明， 膜结合蛋白在转移性侵袭中至关重要， 细胞这些研究表明，酪氨酸激酶及其膜结合 底物高度升高与侵袭性伪足有关， 在侵入性细胞的前沿的细胞突起。形成 侵入伪足和随后的细胞侵入被染料木黄酮阻断， 酪氨酸激酶抑制剂。侵入伪足和脱落膜囊泡 人恶性黑色素瘤细胞系LOX以及膜囊泡 来自实验模型中生长的LOX是一个丰富的来源， 侵袭相关抗原。抗原制备的起始物料为 已经可以分离与侵袭相关的酪氨酸磷酸化的 蛋白质和几种酪氨酸磷酸化蛋白质已经被鉴定 在分离的侵入伪足膜部分中使用现有的抗体。这 建议的工作将：1）分离和微序列的主要未确定的 来自侵袭伪足和脱落膜的酪氨酸磷酸化多肽 LOX细胞的囊泡和LOX肿瘤的膜，使用抗- 磷酸酪氨酸单克隆抗体，和2）鉴定现有的和 产生新的单克隆抗体， 酪氨酸磷酸化蛋白质。蛋白质测序和单克隆 这里获得的抗体将使我们能够识别酪氨酸磷酸化的 专门定位于侵入性皱褶的蛋白质， 与侵入伪足的结合可能形成控制的分子基础 肿瘤细胞的侵袭和转移。

Invasion of tumor cells through basement membranes and interstitial tissues allows tumors to metastasize and colonize new sites in the body. However, the manner in which cell invasion is regulated is completely unknown. Recent studies indicate that tyrosine phosphorylation of membrane-bound proteins is critically important in metastatic invasion of cells. These studies show that tyrosine kinases and their membrane-bound substrates are highly elevated in association with invadopodia, the cellular protrusions at the leading edge of invasive cells. Formation of invadopodia and subsequent cell invasion were blocked by genistein, an inhibitor of tyrosine kinases. Invadopodia and shed membrane vesicles of the human malignant melanoma cell line LOX, as well as membrane vesicles derived from LOX grown in experimental models are a rich source of invasion-related antigens. Starting material for antigen preparation is already available to isolate invasion related-tyrosine phosphorylated proteins and several tyrosine phosphorylated proteins have been identified in isolated invadopodia membrane fractions using existing antibodies. This proposed work will: 1) isolate and microsequence the major unidentified tyrosine phosphorylated polypeptides from invadopodia and shed membrane vesicles of LOX cells, and membranes of LOX tumors using anti- phosphotyrosine monoclonal antibodies, and 2) identify existing and generate new monoclonal antibodies against these invasion-associated tyrosine phosphorylated proteins. Protein sequencing and monoclonal antibodies obtained here will allow us to identity tyrosine phosphorylated proteins that localize exclusively to invasive ruffles and whose association with invadopodia may form the molecular basis for the control of tumor cell invasion and metastasis.