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Identification and Characterization of X-Ray-induced Proteins in Human Cells¹

Division of Cell Growth and Regulation and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana-Farber Cancer Institute (D-810A), Boston, Massachusetts 02115

In order to investigate the biochemical events involved in potentially lethal DNA damage repair (PLDR), we have identified a pleiotropic protein expression response that is activated upon X-irradiation of confluence-arrested human malignant melanoma (U1-Mel) cells. Plateau-phase U1-Mel cells were selected because of their extraordinary capacity for PLDR. Eight major X-ray-induced polypeptides (XIPs) of M_r 126,000–275,000 (i.e., XIP126 through XIP275) were detected by resolving L-[³⁵S]methionine-labeled whole cell extracts using two-dimensional gel electrophoresis. XIPs were found in unirradiated, proliferating U1-Mel cells, shut off under plateau-phase conditions and resynthesized in response to X-irradiation.

The expression of three classes of proteins was affected by X-irradiation. Class I proteins, XIP145 and XIP269, were induced linearly with increasing X-ray doses. The rate of synthesis of class II proteins, XIP126, XIP135, XIP138, XIP141, XIP147, and XIP275, increased linearly with low X-irradiation doses, but plateaued at doses of 150–250 cGy. In contrast, the expression of class III proteins, 47,000 and 254,000 M_r proteins, decreased with increasing X-ray doses.

Tumor, cancer-prone, and normal human cells, which represent a wide range of cells with varied repair capacities, were investigated to better understand the role of XIPs in DNA damage responses. X-irradiated normal and tumor cells induced the synthesis of XIP145 and XIP269. A strong correlation between the induction of XIP269 and PLDR capacity, as measured by delayed plating of plateau-phase cells, was noted. XIP269 was present in six of seven normal and tumor cell types, but was completely absent in cells from patients with Bloom's syndrome and ataxia telangiectasia. X-irradiated Fanconi's anemia and xeroderma pig-mentosum cells synthesized low levels of XIP269. The majority of XIPs synthesized by X-irradiated cells from cancer-prone patients were of low molecular weights.

A number of XIP expression characteristics suggest their role in either gross chromosomal PLDR and/or in X-ray adaptivity responses: (a) XIP expression was inhibited by 1 µg/ml cycloheximide, a dose which decreased survival 6-fold during PLDR holding and resulted in >80% inhibition of protein synthesis; (b) XIP expression was specific for ionizing radiation damage, since heat shock, hypoxia, and alkylating agents failed to induce their synthesis; (c) the time course of expression was long, with the first appearance of XIPs at 3 h and maximal expression at 4 h.

¹ Supported by Grant CA 22427 to Arthur B. Pardee, Ph.D., from the National Cancer Institute, a Radiological Society of North America seed grant to E. N. H., and Biomedical Research Support Grant and Training Grant 5T32 CA 09361 to D. A. B.

² To whom requests for reprints should be addressed.

³ Present address: Department of Radiation Oncology, Hospital of the University of Pennsylvania, Philadelphia, PA 19104.

Received 11/15/88. Revised 2/24/89. Accepted 3/ 6/89.

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