METASTASIS INHIBITION BY FIBRONECTIN-TUMOR INTERACTIONS

我们的结果表明黑色素瘤细胞表面肝素的重要作用 硫酸盐蛋白多糖(HSPG)介导精氨酰-甘氨酰-天冬氨酸(RGD)- 33kD羧基末端肝素与细胞的独立黏附 血浆纤维连接蛋白(FN)A链片段。这得到了以下几个方面的支持 在我们最初的资助期内提出的意见。首先，HSPG， 从体外代谢标记的肿瘤细胞中分离出来，结合到 以浓度依赖的方式涂覆33kD片段的底物， 饱和的时尚。其次，HSPG与该片段的结合是特定的 被产生的细胞黏附抑制单抗(MAb)消除 抗33kD片段。第三，我们鉴定了新的细胞 来自33kD片段的促进黏附的合成肽，也 结合~3H-肝素和黑色素瘤细胞表面HSPG。此外，细胞 这些合成肽的黏附促进活性可以被特异性地 外源性肝素和肝素硫酸糖胺聚糖的抑制作用 (笑话)。这些被命名为I和II的多肽具有主要序列 YEKPGSPPREVVPRPRPGV和KNNQKSEP-LIGRKKT(McCarthy，1988b)。 我们也有证据表明α4β1整合素参与了 FnA链这一区域的细胞识别。这在一定程度上是基于 最近发表的结果(Wayner等人，1989)展示了 α4β1整合素与另一种细胞黏附的相互作用 该片段中的启动子序列称为CS1 (DELPQLVTLPHPNLHPGPEILDVPSKT；Humphries，1987)。与多肽I和II不同， 多肽CS1不能结合~3H-肝素，仅在血浆FNA-1中表达。 锁链。尽管交叉竞争实验表明多肽I、II 和CS1通过不同的分子促进小鼠黑色素瘤细胞黏附 机制，我们的初步结果表明，特定的阿尔法4β 1单抗还抑制细胞与肝素结合肽I和II的黏附， 提示多肽I、II和CS1代表更大的 可与α-4和β-1结合的完整FNA链上的活性部位 整合素和细胞表面HSPG。潜在的、核心的假说 在这些研究中要测试的是，这三个地点，在较大的 结构域，推动HSPG和α4β1整合素在 细胞表面黑色素瘤细胞黏附到这一区域 FN A链。我们将使用特定的抗整合素和抗HSPG单抗，在 与其他试剂，如合成肽和限制性的 含有该结构域的重组FN片段，以证实或驳斥 源自这一假设的预测。具体的比较将是 高转移性黑色素瘤细胞和低转移性黑色素瘤细胞 试图确定细胞黏附的具体变化 与侵袭或转移行为相关的表型。

Our results indicate an important role for melanoma cell surface heparin sulfate proteoglycans (HSPG) in mediating arginyl-glycyl-aspartyl (RGD)- independent cell adhesion to the 33kD carboxyl-terminal heparin binding fragment of plasma fibronectin (FN) A-chains. This is supported by several observations made during our initial funding period. First, HSPGs, isolated from metabolically-labelled tumor cells in vitro, bind to substrate coated with the 33kD fragment in a concentration dependent, saturable fashion. Secondly, HSPG binding to this fragment is specifically abolished by a cell adhesion-inhibiting monoclonal antibody (MAB) generated against the 33 kD fragment. Thirdly, we have identified novel cell adhesion-promoting synthetic peptides from the 33 kD fragment which also bind 3H-heparin and melanoma cell surface HSPG. Furthermore, the cell adhesion promoting activity of these synthetic peptides can be specifically inhibited by exogenous heparin and heparin sulfate glycosaminoglycans (GAGs). These peptides, termed I and II, have the primary sequences YEKPGSPPREVVPRPRPGV and KNNQKSEP-LIGRKKT, respectively (McCarthy, 1988b). We also have evidence for the involvement of an alpha 4 beta 1 integrin in cell recognition of this region of FN A-chains. This is based, in part, on recently published results (Wayner, et al, 1989) demonstrating a role for the interaction of alpha 4 beta 1 integrin with another cell adhesion promoting sequence within this fragment, termed CS1 (DELPQLVTLPHPNLHPGPEILDVPSKT; Humphries, 1987). Unlike peptides I and II, peptide CS1 fails to bind 3H-heparin and is only expressed in plasma FN A- chains. Although cross competition experiments suggest that peptides I, II and CS1 promote murine melanoma cell adhesion by distinct molecular mechanisms, our preliminary results demonstrate that specific alpha 4 beta 1 MABs also inhibit cell adhesion to heparin binding peptides I and II, suggesting that peptides I, II, and CS1 represent portions of a larger active site on intact FN A-chains which can bind both alpha 4 beta 1 integrins and cell surface HSPG. The underlying, and central, hypothesis to be tested in these studies is that these three sites, within the larger domain, drive the association of HSPG and alpha 4 beta 1 integrin on the cell surface as a consequence of melanoma cell adhesion to this region of FN A-chains. We will use specific anti-integrin and anti-HSPG MABs, in conjunction with other reagents such as synthetic peptides and restricted recombinant FN fragments containing this domain, to confirm or refute predictions stemming from this hypothesis. Specific comparisons will be made between highly metastatic melanoma cells and poorly metastatic counterparts, to attempt to identify specific changes in cell adhesion phenotype which correlate with invasive or metastatic behavior.