METASTASIS INHIBITION BY FIBRONECTIN-TUMOR INTERACTIONS

我们的研究结果表明黑色素瘤细胞表面肝素的重要作用 硫酸蛋白聚糖（HSPG）介导乙酰-甘氨酰-乙酰（RGD）- 独立的细胞粘附至33 kD羧基末端肝素结合 血浆纤连蛋白（FN）A链片段。 这一点得到了几位 在我们最初的融资过程中。 首先，HSPG， 从体外代谢标记的肿瘤细胞中分离， 用33 kD片段以浓度依赖性包被的底物， 可饱和的方式。 其次，HSPG与该片段的结合是特异性的， 通过产生的细胞粘附抑制单克隆抗体（MAB）消除 与33 kD片段进行比较。 第三，我们发现了新的细胞， 来自33 kD片段的促粘附合成肽，其还 结合3 H-肝素和黑色素瘤细胞表面HSPG。 此外，细胞 这些合成肽的粘附促进活性可以特异性地 被外源性肝素和硫酸肝素糖胺聚糖抑制 （GAGs）。 这些肽，称为I和II，具有一级序列 YEKPGSPPREVPRPRPGV和KNNQKSEP-LIGRKKT（McCarthy，1988 b）。 我们也有证据表明α 4 β 1整合素参与了 细胞识别FN A链的该区域。 这部分是基于 最近发表的结果（Wayner等人，1989年）表明， α 4 β 1整合素与另一种细胞粘附 该片段内的启动序列，称为CS 1 （DELPQLVTLPHPNLHPGPEILDVPSKT; Humphries，1987）。 与肽I和肽II不同， 肽CS 1不能结合3 H-肝素，并且仅在血浆FNA-1中表达。 店 尽管交叉竞争实验表明肽I、II 和CS 1通过不同的分子途径促进小鼠黑色素瘤细胞粘附 机制，我们的初步结果表明，特定的α 4 β， 1 MAB还抑制细胞粘附于肝素结合肽I和II， 这表明肽I、II和CS 1代表了一个更大的 完整FN A链上的活性位点，可结合α 4 β 1 整合素和细胞表面HSPG。 基本的核心假设是 在这些研究中要测试的是，这三个网站，在较大的 结构域，驱动HSPG和α 4 β 1整合素在 黑色素瘤细胞粘附到细胞表面的这一区域， FN A链。 我们将使用特异性抗整联蛋白和抗HSPG单克隆抗体， 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，以证实或反驳 这一假设所产生的预测。 具体比较将在 在高转移性黑色素瘤细胞和低转移性黑色素瘤细胞之间 对应，试图确定细胞粘附的具体变化， 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.