MOLECULAR BASIS OF MICROENVIRONMENTAL CELL CYCLE CONTROL

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肿瘤生物学中尚未解开的主要谜团之一是体内肿瘤细胞以可逆方式退出细胞周期的机制。经过几十年的研究，一个隐含的假设是，营养供应的限制，无论是通过有限的渗透到细胞团中，还是限制流向局部区域的血液，都会创造一个应激的微环境，导致细胞停止其细胞周期的传递。由于这种机制研究的实验肿瘤的众所周知的局限性，我们使用多细胞肿瘤球体模型来进行这个项目的大部分。球体非常适合于这样的研究，这既是因为它们对微环境和细胞增殖梯度的对称排列，也是因为我们在实验上利用这种对称性的独特能力。具体地说，我们可以从椭圆形微环境中的已知位置分离完整的、有活力的细胞，以详细研究与细胞周期停滞相关的分子变化。为了提供体外系统和体内情况之间的联系，我们将确定我们提出的机制是否适用于实际的肿瘤。我们追求四个具体目标：1)确定多细胞球体细胞周期停滞的分子基础；2)确定是否同样的分子机制在体内肿瘤中起作用；3)确定诱导球体细胞周期停滞的微环境信号(S)；以及4)确定辐射和微环境诱导的细胞周期停滞之间的相互作用。流动分析既用于常规的DNA含量分析，也用于通过双标记DNA分析技术确定溴脱氧尿苷的摄取。这些细胞周期数据对于与我们的分子分析进行比较至关重要。我们还在利用荧光标记的抗体通过Flow来测量细胞周期蛋白和细胞周期蛋白依赖性蛋白激酶的表达。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One of the major unsolved mysteries in tumor biology is the mechanism by which tumor cells in vivo exit from the cell cycle in a reversible fashion. An implicit assumption through decades of research is that limitations of nutrient supply, either by limited penetration into the cell mass or restricted blood flow to local regions, create a stressful microenvironment which induces cells to arrest their cell cycle transit. Due to well-known limitations of experimental tumors for such mechanistic studies, we are using the multicellular tumor spheroid model for the majority of this project. Spheroids are ideally suited for such studies, both because of their symmetrical arrangement of microenvironmental and cellular proliferation gradients, and because of our unique ability to experimentally exploit this symmetry. Specifically, we can isolate intact, viable cells from known locations within the spheroid microenvironment for detailed study of the molecular changes associated with cell cycle arrest. In order to provide a link between this in vitro system and the in vivo situation we will determine whether our proposed mechanism is operative in actual tumors. We are pursuing four Specific Aims: 1) to determine the molecular basis for cell cycle arrest in multicellular spheroids; 2) to determine if the same molecular mechanisms are operative in tumors in vivo; 3) to identify the microenvironmental signal(s) which induce cell cycle arrest in spheroids; and 4) to determine the interaction between radiation- and microenvironmentally-induced cell cycle arrest. Flow analysis is used both for routine DNA content analysis, and also for determining the uptake of bromodeoxyuridine by means of a dual-label DNA analysis technique. These cell cycle data are critical for comparison with our molecular analysis. We are also pursuing the measurement of cyclin and cyclin-dependent kinase expression by flow using fluorescently-tagged antibodies.