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

D. A. Boothman, I. Bouvard, E. N. Hughes

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Abstract

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 (Ul-Mel) cells. Plateau-phase Ul-Mel cells were selected because of their extraordinary capacity for PLDR. Eight major X-ray-induced polypeptides (XIPs) of Mt 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 Ul-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 Mr 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 pigmentosum 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 4h.

Original languageEnglish (US)
Pages (from-to)2871-2878
Number of pages8
JournalCancer research
Volume49
Issue number11
StatePublished - Jun 1 1989

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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    Boothman, D. A., Bouvard, I., & Hughes, E. N. (1989). Identification and Characterization of X-Ray-induced Proteins in Human Cells. Cancer research, 49(11), 2871-2878.