[CANCER RESEARCH 51, 5587-5595. October 15. 1991] Induction of Tissue-type Plasminogen Activator by Ionizing Radiation in Human Malignant Melanoma Cells1 David A. Boothman,2 Meizhi Wang, and Sam W. Lee Department of Radiation Oncology. Division of Cancer Biology, University of Michigan, Ann Arbor, Michigan 48109 [D. A. B., M. H'J, and Dana-Farher Cancer Institute, Division of Cancer Genetics, Harvard University, Boston, Massachusetts 02115 [S. H'. L.] ABSTRACT Two differently timed extracellular and intracellular enzymatic and mRNA peaks of tissue-type plasrninogen activator (t-PA) were induced following ionizing radiation. The first peak appeared within 10 min following \-irradiation but rapidly declined. The appearance of early t- PA mRNA transcripts and enzymatic activity were not prevented by actinomycin D treatment. In contrast, cycloheximide prevented the early, minor enzymatic induction peak of t-PA. Stabilization of t-PA mRNA transcripts appears to be an early initial response of human cells to ionizing radiation, since the synthesis of new mRNA transcripts within the first 30 min was not observed via nuclear run-on analyses. Nearly 12 h following \-irradiation, a second major enzymatic peak of t-PA was observed. Cycloheximide or actinomycin D treatments blocked the later t-PA response. t-PA mRNA levels were induced > 100- fold in 4 h by ionizing radiation as assayed via Northern or nuclear run- on analyses. During the major induction period, t-PA mRNA transcripts reached their maximum levels at 4-8 h, and intracellular enzyme levels accumulated 6-8 h after X-irradiation. Unirradiated Ul-Mel cells dem onstrated only low basal levels of t-PA mRNA and enzymatic activity. Similar induction responses were found following UV-irradiation or 12- O-tetradecanoyl-phorboI-13-acetate (I'M A) treatments. Normal human fibroblast (i.e., GM 2936B, GM2907A, and IMR-90) cells also demonstrated the induction of t-PA, although only one later enzymatic peak was detected. The induction of t-PA mRNA levels and intracellular and extracellular enzymatic activities for these cells were 50-fold lower than with Ul-Mel cells given equitoxic doses of X-rays. Differential expression of t-PA in some tumor as compared to normal tissues may be utilized in future chemotherapeutic regimens. INTRODUCTION Several laboratories have reported the induction of various gene products in mammalian cells by a wide range of agents that cause cytotoxic stress (see Refs. 1-3 for reviews). The regulation of new gene transcripts and gene products following various DNA insults now include: (a) DNA binding transcrip tion factors, such as the UV-responsive element binding factors and X-ray-induced protein 175 (XIP175 bp) (1, 4-6); (b) pro- tooncogenes, such as c-fos (7), c-jun (8), and c-myc (9); (c) several growth-related genes, such as PCNA (10), protein kinase C (11), interleukin-1 (11), and p53 (12); and (d) a variety of other genes, including proteases (13, 14), RNA polymerase-ß (15), tumor necrosis factor (16), metallothioneins (17, 18), and DT Diaphorase (19).' These gene products may influence later events within damaged mammalian cells, including mutagene- sis, carcinogenesis, metastasis, cell survival and repair, and cell Received 2/22/91; accepted 8/8/91. 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 a grant from the American Cancer Society (PDT- 412). 'To whom requests for reprints should be addressed, at Department of Radiation Oncology, Division of Cancer Biology, University of Michigan. 1331 East Ann Street. Rm 4131, Ann Arbor. MI 48109. J D. A. Boothman, S. W. Lee, M. Wang, and E. N. Hughes. Isolation and partial characterization of X-ray-inducible gene transcripts from radioresistant human melanoma cells, in preparation, 1991. Presented at the 82nd annual meeting of the American Association for Cancer Research, abstract 1671, p. 281. Houston, TX, 1991. death (i.e.. apoptosis) (1,3, 20-22). Using differential hybridization, we have recently isolated 12 cDNA4 clones which were dramatically induced by ionizing radiation; the results and specific details of the differential hybridization screen will be described elsewhere.4 After the clones were isolated, we partially sequenced the cDNA inserts, and their identities (if known) were elucidated via GenBank sequence analyses.1 One of the cDNA clones isolated contained >95% DNA sequence homology to human tissue-type plasrnin ogen activator. t-PA is a secreted serine glycoprotease of approximately M, 70,000, which is produced by various different cell types and tissues and by many human tumor cells in culture (23-26). Both t-PA and its related serine protease, u-PA, participate in the regulation of a multitude of cellular processes leading to the formation of active plasmin, which then initiates a number of cascade reactions. These proteases are therefore thought to be important in circulatory fibrinolytic activity and may act in concert to initiate prohormone processing, inflammation, re production, and cell migration, including an involvement in tumor cell formation and metastasis (see Ref. 23 for review), t- PA has been localized to chromosome 8, and its cDNA and complete genomic sequence have been reported (27-29). Plasminogen activators are regulated at the enzymatic level by various external stimuli. Increased levels of u-PA and/or t- PA can be induced by hormones or hormone-like agents in distinct tissue-specific manners (25, 30, 31). The induction of t-PA has been associated with the intracellular induction of protein kinase C (32). Increased secretion of u-PA enzymatic activity has been reported with various mammalian cells in response to exposure to a limited number of alkylating agents (13, 14, 33). The induction of t-PA by ionizing radiation has not been previously demonstrated, nor has the induction/sta bilization of t-PA been investigated at the level of transcription following any DNA-damaging agent. The purpose of the present study was to further investigate the induction of t-PA by ionizing radiation. The temporal induction of enzymatic extra- and intra-cellular levels of t-PA and the regulation of its mRNA were followed after exposure of confluence-arrested Ul-Mel cells to various doses of ionizing radiation and after exposure to other DNA-damaging agents. We also compared the induction of t-PA in Ul-Mel cells to that induced by density-arrested normal human fibroblasts (i.e., GM2936B, GM2907A, and IMR-90). 4 The abbreviations used are: cDNA, complementary DNA; Ul-Mel, radiore sistant human malignant melanoma; t-PA, tissue-type plasminogen activator; u- PA, urokinase-type plasminogen activator; PMA, 12-O-tetradecanoyl-phorbol- 13-acetate:f!-PKC, beta-protein kinase C; PLDR, potentially lethal DNA damage repair. 5 N. A. Fukunaga, R. A. Schea, H. L. Burrows, and D. A. Boothman. Enhanced induction of tissue-type plasminogen activator in human cancer-prone as opposed to normal cells, in preparation. 1992. Presented at the Miami Winter Symposium, Miami, FL, Feb., 1991. 5587 on March 15, 2016. © 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from