(CANCER RESEARCH 50, 5045-5048, August 15, 1990] Differential Effect of Ionizing Radiation on Transcription in Repair-deficient and Repair-proficient Mice1 George P. Munson2 and Gayle E. Woloschak3 Biological and Medical Research Division, Argonne National Laboratory, Argonne, Illinois 60439 ABSTRACT Experiments were designed to examine in vivo changes in total tran scription and in the expression of the c-fos gene following whole-body exposure of mice to JANUS fission-spectrum neutrons. Radiation repair- deficient (wst/wst) and -proficient (wst/9, C57BL/6 x C3H F,) mice were exposed to JANUS fission-spectrum neutrons calibrated to deliver a gut dose of 50 cGy. Animals were sacrificed <10 or at 60 min postirradiation, and gut tissues were removed for study. Our results revealed that, in repair-proficient mice, an immediate depression (relative to untreated controls) in total transcription was evident that continued through lh postirradiation. Conversely, radiation-sensitive wst/wst mice displayed doubled transcription levels postirradiation. Expression of c-fos was consistently depressed following radiation exposure in control and wst/ tvst mice. However, the depression of c-fos mRNA was delayed in wst/ wst mice relative to controls. These results demonstrate abnormal regu lation of transcription and of c-fos mRNA accumulation in repair-deficient wasted mice following exposure to ionizing radiation. In addition, this work documents rapid total transcriptional depression in normal mice following radiation exposure. INTRODUCTION Recent work elucidating the transcriptional effects of radia tion delivered to cells in culture has been reported by us (1) and others (2-4). In this paper, experiments were designed to deter mine whether observed in vitro effects of radiation-induced damage can be detected in vivo as well. Specifically, we were interested in determining whether total transcription is re pressed and whether c-fos mRNA accumulation is induced following whole-body exposure to ionizing radiations, as has been reported previously following radiation exposure of cells in culture (1-4). The experiments reported here then were designed to measure total transcription and gene-specific (c- fos) mRNA accumulation in the gut of mice following exposure in vivo to 50 cGy of JANUS fission-spectrum neutrons, c-fos was chosen for study because of its normally high expression in gut tissue and its reported induction following in vitro ex posure to DNA-damaging agents (3). Concomitant to studies with control mice, we also examined neutron-mediated transcriptional responses in radiation-sensi tive wasted (wst/wst) mice. Mice bearing the autosomal recessive mutation wst develop a disease similar to human ataxia telan- giectasia with manifestations that include low immune re sponses at secretory sites, neurological abnormalities, and in creased sensitivity of lymphocytes to the lethal effects of ion izing radiation (5-7). The radiation sensitivity of cells from wasted mice is characterized by increased chromosomal abnor malities in bone marrow cells following exposure to radiation Received 11/2/89; revised 4/27/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by the US Department of Energy, Office of Health and Environmental Research, under Contract W-31-109-ENG-38. 2 Present address: Dept. of Medicine, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153. Work was done while a student in the laboratory of G. E. W. 3To whom requests for reprints should be addressed. (5) and increased sensitivity of T-cells to the lethal effects of y- rays.4 The studies reported here indicated that, in control mice, exposure to radiation was accompanied by an immediate repres sion in total transcription that continued for at least l h following irradiation. In radiation-sensitive wasted mice, how ever, total transcription doubled following similar neutron ex posure. Expression of c-fos mRNA accumulation was consist ently depressed by radiation exposure in both wasted and con trol mice, although this depression took longer to develop in radiation-sensitive wasted mice. These results demonstrate that some responses are similar in vitro and in vivo following radia tion exposure (such as total transcription inhibition), while others differ (c-^òs-specificmRNA accumulation). MATERIALS AND METHODS Mice. Breeding pairs of heterozygous wst/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice used in these experiments were bred in the animal facilities at Argonne National Laboratory. Littermates of known wst/wst mice (determined phenotyp- ically) were designated as tvst/9. Mice designated wst/9 should consist of both wst/+ (67%) and wild-type +/+ (33%). Age-matched C57BL/ 6 x C3H F, (hereafter called B6C3F,) mice were included in this study because previous work had suggested that wst/+ hétérozygotes may express some abnormalities, even though this group cannot be pheno- typically distinguished from the +/+ wild-type (7). All mice used in these experiments were 26 to 28 days old. (The wst defect is first evident at 21 days of age, and mice die by 28 to 31 days.) Each data point represents results from tissues of 3 to 5 mice pooled; all experiments were repeated 3 times. Irradiation. Mice were irradiated in the JANUS reactor at Argonne National Laboratory, which was calibrated to deliver a total gut dose of 50 cGy of fission-spectrum neutrons at a dose rate of 12 cGy/min. Irradiated mice were sacrificed by asphyxiation immediately or lh postirradiation, and gut tissues (small and large intestines) were re moved and placed on ice for further study. Total Transcription Assay. Nuclei (from gut tissues) were isolated as described (8, 9). In brief, cell suspensions were separated by low-speed (2000 x g) centrifugation through a 1 M sucrose pad. On the basis of DNA content as determined by spectrofluorimetry, nuclei were ali- quoted in equal numbers and frozen at -70°C.For transcription assays, equal numbers of nuclei were incubated for 30 min at room temperature in a reaction mixture containing [a-32P]UTP (8, 9). DNase I was then added, and samples were incubated an additional 30 min at room temperature. Nucleic acids were then precipitated from cold 10% trichloroacetic acid and isolated on glass fiber filters under vacuum. Relative percentages of total transcription in cells from treated mice were determined by comparisons to specific activity incorporated into RNA from untreated cells. RNA Preparation. Gut tissues (small and large intestines) were homogenized in equal volumes of homogenization buffer (75 ITIM NaCl:20 mM Tris:2 ITIMEDTA, pH 8.0) and phenol. Nucleic acids were precipitated from 3 M sodium acetate, pH 6.0, at 4°Cand then from ethanol at -20°C(8, 9). Total RNA to be used in dot blots was digested with RNase-free DNase I (Promega Biotec) extracted from 5045 4 M. Padilla. C. R. Libertin, C. J. Krco, and G. E. Woloschak. Radiation sensitivity of T-cells from wasted mice. Cell. Immunol., in press. 1990. Research. on October 25, 2021. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from