X RAY INACTIVATION OF HUMAN COLORECTAL CANCER CELLS

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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.