X RAY INACTIVATION OF HUMAN COLORECTAL CANCER CELLS

在待研究的等基因细胞系中，p53、p21或 14-3-3要么完好无损，要么已被删除。然后，随后的分子和 生化研究可以通过以下方式集中于了解这些机制 三种不同基因表达的哪一种变化会影响 特定的辐射诱导的细胞改变 在细胞周期的不同阶段受到辐射。计算机化视频延时 (CVTL)将被利用，以便单个细胞及其后代可以 追踪足够长的时间以确定每个受辐射的细胞是克隆性的还是 非克隆性。由于单个细胞在照射后被跟踪，因此 将确定导致细胞死亡的特定变化。这个 申请人假设p53、p21和14-3-3的表达对两者都有影响 X射线照射后的细胞周期进程和细胞完成的能力 正常的除法。他进一步假设，这些效应改变了模式 当细胞受到辐射时，细胞死亡和辐射敏感性的变化 在细胞周期的不同阶段。重组蛋白14-3-3的表达 据报道，辐射将细胞周期蛋白B1/CDC2隔离在细胞质中，并且 从而防止它移位到必须被激活的原子核 CDC25C存在于细胞质中，以使细胞进入有丝分裂。申请人 然后假设14-3-3等位基因的缺失将减少辐射 诱导G2延迟和受照细胞成功完成 有丝分裂。将通过比较所获得的结果来进行CVTL研究 与亲本细胞系HCT116(p53+/+，p21+/+，14-3-3+/+)进行比较，结果 用四种等基因敲除衍生物(p53-/-、p21-/-、14-3-3-/-)获得 和p21-/-14-3-3-/-)双基因敲除系，由Dr. 沃格尔斯坦。细胞周期进程将根据CVTL序列进行量化 相位对比度和荧光图像。申请者还计划测试一个 建议的模型中，细胞周期蛋白B1、CDC2、CD25C和p21的水平将是 确定为细胞在经历任一细胞死亡之前在G2中延迟， 不成功的异常有丝分裂或成功的正常分裂。

In isogenic cell lines to be studied, both alleles of p53, p21, or 14-3-3 are either intact or have been deleted. Then, subsequent molecular and biochemical studies can be focused towards understanding the mechanisms by which changes in the expression of the three different genes affects the particular radiation-induced cellular alterations induced in cells that are irradiated in different phases of the cell-cycle. Computerized video time lapse (CVTL) will be utilized so that individual cells and their progeny can be followed long enough to determine if each irradiated cell is clonogenic or non-clonogenic. As the individual cells are followed after irradiation, the particular alterations that result in cell death will be identified. The applicant hypothesizes that the expression of p53, p21, and 14-3-3 affects both cycle progression after X-irradiation and the ability of the cells to complete a normal division. He further hypothesizes that these effects alter the modes of cell death and variations in radio-sensitivity when the cells are irradiated in different phases of the cell cycle. The expression of the protein 14-3-3 by radiation has been reported to sequester cyclin B1/Cdc2 in the cytoplasm, and thus keep it from translocating to the nucleus where it must be activated by Cdc25C in the cytoplasm in order for the cell to enter mitosis. The applicant then hypothesizes that deletion of the 14-3-3 alleles will reduce the radiation induced G2 delay and ability of the irradiated cell to complete a successful mitosis. The CVTL studies will be carried out by comparing results obtained with the parental cell line, HCT116 (p53+/+, p21+/+, 14-3-3+/+) with results obtained with the four isogenic knockout derivatives (p53-/-, p21-/-, 14-3-3-/- and p21-/- 14-3-3-/-) double knockout lines, which were supplied by Dr. Vogelstein. Cell-cycle progression will be quantified from CVTL sequential phase contrast and fluorescence images. The applicant also plans to test a proposed model, in which levels of cyclin B1, Cdc2, Cdc25C, and p21 will be determined as cells are delayed in G2 prior to undergoing either cell death, an unsuccessful abnormal mitosis, or a successful normal division.