X RAY INACTIVATION OF HUMAN COLORECTAL CANCER CELLS

在待研究的同基因细胞系中，p53、p21或p23的两个等位基因均被检测到。 14-3-3要么是完整的，要么已经删除。然后，随后的分子和 生物化学研究可以集中在了解机制， 这三种不同基因表达的变化会影响 在细胞中诱导的特定辐射诱导的细胞改变， 在细胞周期的不同阶段照射。计算机化视频延时 将利用CVTL，使得可以分离单个细胞及其后代。 跟踪足够长的时间以确定每个照射的细胞是否是克隆形成的或 非克隆性的。当照射后跟踪单个细胞时， 将鉴定导致细胞死亡的特定改变。的 申请人假设p53、p21和14-3-3的表达影响这两者 X射线照射后的周期进展和细胞完成 正常的分裂。他进一步假设这些效应改变了 当细胞受到辐射时， 在细胞周期的不同阶段。蛋白质14-3-3的表达， 据报道，辐射可将细胞周期蛋白B1/Cdc 2隔离在细胞质中， 从而防止它转移到核中，在那里它必须被激活， Cdc 25 C在细胞质中的存在，以便细胞进入有丝分裂。申请人 然后假设14-3-3等位基因的缺失将减少辐射 诱导的G2延迟和照射细胞完成成功的细胞周期的能力。 分裂。CVTL研究将通过比较获得的结果进行 与亲本细胞系HCT 116（p53+/+、p21+/+、14-3-3+/+）进行比较，结果 用四种同基因敲除衍生物（p53-/-，p21-/-，14-3-3-/-）获得的 和p21-/-14-3-3-/-）双敲除系，其由Dr. 沃格尔斯坦将从CVTL连续定量细胞周期进展 相位对比和荧光图像。申请人还计划测试一个 提出的模型，其中细胞周期蛋白B1，Cdc 2，Cdc 25 C和p21的水平将是 确定为细胞在经历细胞死亡之前延迟于G2， 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.