[CANCER RESEARCH 52. 3814-3817. July 1. I992| Advances in Brief Gadd45 and Gaddl53 Messenger RNA Levels Are Increased during Hypoxia and after Exposure of Cells to Agents Which Elevate the Levels of the Glucose- regulated Proteins1 Brendan D. Price2 and Stuart K. Calderwood Stress Protein Group, Joint Center for Radiation Therapy, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 Abstract We have investigated overlapping activation pathways for two families of stress genes that are expressed in cells exposed to hypoxia. The growth arrest and DNA damage (gadd) genes are induced by DNA damage and irradiation, and their expression is associated with growth arrest. The glucose-regulated proteins (GKPs) are induced by chemical agents that disrupt protein trafficking in the endoplasmic reticulum such as tunica- mycin and A23187 and by hypoxia. Here, we demonstrate that the treatment of NIH-3T3 cells with chemical inducers of GRPs results in increased levels otgadd45 and gadd153 mRN A as well as GRP78 mRNA. In addition, hypoxia was also able to increase gadd45, gaddl53, and CRP78 mRNA. Therefore the GRP and gadd genes can be activated by similar stimuli (e.g., hypoxia and chemical inducers). However, the mechanisms leading to increased levels of GRP78 and gadd gene mRNA are different and may involve distinct protein kinases. Increased expres sion of GRPs after treatment with chemical inducers is sensitive to cycloheximide and the protein kinase inhibitors genistein, 2-aminopurine, and 117, whereas the increase in gadd gene mRNA could be blocked by the protein kinase inhibitors 117 and 2-aminopurine but not by genistein or cycloheximide. GRP78 induction occurs by a pathway that requires protein synthesis and is sensitive to genistein. H7, and 2-aminopurine, whereas gadd gene induction is independent of protein synthesis and is inhibited by 117 and 2-aminopurine only. Introduction The gadd' genes are induced by growth arrest, by DNA damage, or by X-ray or UV irradiation (1-3). One member of the gadd gene family, gadd45, is strongly induced by low doses (2 Gy) of X-ray irradiation by a mechanism which does not involve activation of protein kinase C (4). One of the main effects of the radiation of cells is to induce growth arrest, with cells being blocked in G2 (6, 7). This block is thought to allow cells time to repair DNA damage before proceeding to cell division (6, 7). Cells which are forced to proceed past this block without allowing time for DNA repair (by treating the cells with caffeine) exhibit reduced cell survival and DNA fragmen tation (7). In yeast, the rad9 gene is thought to be responsible for blocking cells in G? after irradiation (6). The gadd genes may encode mammalian proteins with functions similar to that of the rad9 gene, although they have no significant sequence homology to the rad9 gene (3, 4). gadd45 has 60% homology to MyDIlS, a gene induced in murine myeloblastic leukemia cells during terminal differentiation and growth arrest (5). Thus the induction of the gadd genes by radiation and/or DNA- Received 5/6/92; accepted 5/27/92. 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. ' Supported by NIH Grants R29CA44940 and ROÕ47407. 2To whom reprint requests should be addressed, at Dana-Farber Cancer Institute, 44 Binney Street, Room JF205. Boston. MA 02115. 1The abbreviations used are: gadd. growth arrest and DNA damage: GRP. glucose-regulated proteins; ER. endoplasmic reticulum. damaging agents may be required to growth arrest the cells and allow time for DNA repair. We were interested in how varia tions in the environment of the cell might affect the expression of the gadd genes. Hypoxia has been shown, using tissue culture systems, to confer radioresistance on cells (8), and hypoxic cells have been detected in tumors (9). However, there has been little study on how environmental conditions such as hypoxia may affect gene expression and how this may affect the response of cells to irradiation. The best-studied gene family whose tran scription is altered by hypoxia is the GRPs. GRPs are major protein constituents of the ER (10-13). GRP78 functions as a molecular chaperone in the ER, where it participates in the folding of nascent proteins and the formation of oligomeric proteins (14). When cells are deprived of glucose (12), become hypoxic ( 11), or are treated with stress compounds that interfere with ER function (e.g., tunicamycin, A23187, thapsigargin)(12, 13, 15), the transcription of the GRPs is greatly increased. These stress agents may all act by inhibiting the glycosylation of proteins in the ER, which results in the retention of proteins within the lumen of the ER (10-14). This accumulation of proteins may be the stimulus for enhanced GRP transcription. Here, we show that both hypoxia and chemical inducers of GRP expression can cause increased expression of the gadd genes. However, the induction of the gadd genes apparently occurs by a different pathway to the induction of GRPs, since the two gene families showed differential sensitivities to protein synthesis inhibitors (cycloheximide) and protein kinase inhibi tors (genistein, 2-aminopurine, H7). Materials and Methods Cell Culture, Inhibitor Treatment, and Hypoxia. NIH-3T3 cells were grown to about 90% confluency in Dulbecco's modified Eagle's medium containing 10% bovine calf serum. Inhibitors (obtained from Sigma Chemical Co., St. Louis, MO) were added at time zero and remained in the medium throughout the experiment unless otherwise stated. Cells were exposed to hypoxia as follows. NIH-3T3 cells were overlain with 10 ml of fresh Dulbecco's modified Eagle's medium/10% serum (glucose = 4.5 g/dtrr'), sealed in air-tight chambers, and maintained at 37°Cthroughout the subsequent manipulations. The 95% air/5% CO2 medium was removed by flushing the chambers with 95% nitrogen/ 5% CO2, after which the concentration of oxygen was calculated to be 67 ppm (16). During hypoxia, approximately 20% of the available glucose was used after 20 h, and the pH of the medium remained close to 7.4 throughout the experiment. Cell viability was monitored with trypan blue; essentially all cells (>96%) excluded the dye at the end of the treatment, except for hypoxic cells incubated in either H7 or 2- aminopurine, 30% of which stained at the end of the incubation. For this reason, the treatment of hypoxic cells with H7 or 2-aminopurine was not studied (see text). Northern Blotting. After the treatment of cells with inducers, cells were washed twice in phosphate-buffered saline, and total cellular RNA was prepared using the guanidinium isothiocyanate method (17). RNA ( 15 /ug) was loaded and separated on 1% agarose/2.2 M formaldehyde 3814 Research. on October 17, 2021. © 1992 American Association for Cancer cancerres.aacrjournals.org Downloaded from