COORDINATE REGULATION OF PROTEINS ASSOCIATED WITH RADIATION-RESISTANCE IN CULTURED INSECT CELLS

Cultured TN-368 lepidopteran insect cells exhibit a pronounced resista nce to the lethal effects of a variety of physical agents, including X rays and 254 nm UV light, as well as a large number of chemicals. The resistance to ionizing radiation has previously been associated with an inducible process which is not expressed in unirradiated cells or c ells receiving less than some minimal amount of radiation necessary fo r activating the process. The studies in this paper were initiated in an attempt to identify and characterize the inducible proteins associa ted with the marked radiation resistance of the TN-368 cells. Cells we re exposed to doses of 0, 25, 64 or 350 Gy of Cs-137 gamma rays and in cubated either for 3 h in medium containing [S-35]methionine or for 2 h without labeling. Labeled cells were separated into nuclear and cyto plasmic fractions and proteins were analyzed on two-dimensional polyac rylamide gels. Unlabeled cells were used to isolate total RNA which wa s translated in vitro in a rabbit reticulocyte lysate system with S-35 label. These translation products were also analyzed by two-dimension al electrophoresis. Gamma irradiation of the TN-368 cells resulted in the de novo synthesis of several proteins as well as the complete inhi bition of others. The number of such proteins identified was 19. These proteins ranged in size from 18-73 kDa, with a pI distribution of 4.7 to 6.1. In addition to the unique proteins, a large number of other p roteins were also either up- or down-regulated. These observations wer e made in both nuclear and cytoplasmic fractions as well as in the tra nslation products of RNA produced after irradiation. These studies ind icate that RNA and protein synthesis in lepidopteran cells are coordin ately regulated in response to ionizing radiation and may participate in the pronounced radioresistance of the TN-368 cells.