[CANCER RESEARCH 56. 3617-362], August 15. I9%| Advances in Brief Nuclear Signaling Induced by Ionizing Radiation Involves Colocalization of the Activated p56/p53lyn Tyrosine Kinase with p34cdc21 Surender Kharbanda, Aliameli Saleem, Zhi-Min Yuan, Stine Kraeft, Ralph Weichselbaum, Lan Bo Chen, and Donald Kufe2 Divisions of Cancer Pharmacology [S. Kh., A. S., Z-M. Y., D. KJ ami Cellular ami Molecular Biology ¡S.Kr.. L B. C.I, Dana-Farher Cancer Institute. Harvard Medical School. Boston. Massachusetts 02115, and Department of Radiation and Cellular Oncolo^\. Pritzkcr School of Medicine. Universi^' of Chicago, Chicago, Illinois f)0f>37¡R.W.j Abstract The Src-like protein-tyrosine kinase p56/p53ly" associates with cell membranes and transduces signals from activated cell surface receptors. In the present work, cell fractionation and confocal microscopy studies demonstrate expression of Lyn in the nucleus. We also demonstrate that exposure of intact cells to ionizing radiation is associated with selective activation of nuclear Lyn. Similar findings have been obtained following irradiation of purified nuclei. Immunoprecipitation studies of nuclear lysates demonstrate radiation-induced binding of Lyn to p.M"1':. Nuclear colocalization of Lyn with Cdc2 has been confirmed by confocal micros copy. Other studies with glutathione S-transferase-Lyn fusion proteins demonstrate that the binding of Lyn to nuclear Cdc2 is associated with inhibition of Cdc2 activity. These findings suggest that the association of activated Lyn with Cdc2 in the nucleus may contribute to regulation of a DNA damage-dependent premitotic checkpoint. Introduction The cellular response to IR3 includes cell cycle arrest, activation of DNA repair, and lethality ( 1). The signaling mechanisms responsible for the regulation of these events, however, remain unclear. Recent studies have shown that exposure of eukaryotic cells to IR is associ ated with activation of serine/threonine protein kinases and the induc tion of certain early response genes that code for transcription factors (2-5). Other studies have shown that levels of the tumor suppressor p53 protein increase during IR-induced arrest of cells in G, (6, 7). IR also activates protein kinase C and PTK activities (3, 8-10). Exposure of bacteria to DNA-damaging agents activates the recA protease and a cascade of events referred to as the SOS response (11, 12). Although DNA damage and arrest of replication forks are believed to initiate the bacterial survival response, studies in mammalian cells exposed to UV light have supported the involvement of Src family PTKs (13). Be cause the Src-like PTKs associate with the inner surface of the cell membrane and transduce signals from activated cell surface receptors (14), these findings have suggested that mammalian cells respond to certain agents with induction of signals from the cell membrane rather than the nucleus. A member of the Src family, p56/p53lyn, contains SH2 and SH3 domains and an amino-terminal sequence, which, when myristoylated, serves as a membrane localization signal (14). In B lymphocytes, Lyn associates with membrane-bound IgM and participates in antigen- mediated signaling (15, 16). Engagement of the B-cell antigen recep- Received 5/22/96; accepted 7/10/96. 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. 1 This investigation was supported by United States Public Health Service Grant CA55241, awarded by the National Cancer Institute. United States Department of Health and Human Services. 2 To whom requests for reprints should be addressed, at Division of Cancer Pharma cology, Dana-Farber Cancer Institute. 44 Binney Street, Boston, MA 02115. 1 The abbreviations used are: IR, ionizing radiation: GST, glutathione S-transferase; PTK. protein-tyrosine kinase: PDGF. platelet-derived growth factor. tor induces activation of Lyn and its association with phosphatidyli- nositol 3-kinase, GTPase activating protein, and phospholipase Cy2 (17). Other studies have demonstrated that Lyn associates with gan- gliosides (18) and glycophosphatidylinositol-anchored cell surface molecules (19, 20). Recent work has demonstrated that exposure of human HL-60 myeloid leukemia cells to IR is associated with acti vation of Lyn and its association with p34cdc2 (10, 21). The activity of p34cdc2 jn a comp|ex wjt|1 CyCi¡n g ¡s required for the G2-M check point (22). Although previous studies have demonstrated that IR exposure is associated with inhibition of p34cdc2 activity as a mech anism of G2 arrest (23), the signaling events that regulate p34cdc2 in irradiated cells remain unclear. The present studies have examined the effects of IR treatment on activation of Lyn in human HL-60 myeloid leukemia cells. Cell fractionation and confocal microscopy studies demonstrate expression of Lyn in the nucleus and the cell membrane. The results further demonstrate activation of nuclear and not membrane Lyn in irradiated cells. We also demonstrate radiation-induced binding of nuclear Lyn to p34cdc2. Materials and Methods Cell Culture. HL-60 myeloid leukemia cells were grown in RPMI 1640 containing 15% heat-inactivated fetal bovine serum supplemented with 100 units/ml penicillin, UK) /xg/ml streptomycin, 2 mM L-glutamine. 1 HIMsodium pyruvate, and 1 mM nonessential amino acids. Cells in logarithmic growth phase were suspended in complete RPMI 1640 with 0.5% fetal bovine serum for 18 h before irradiation. Irradiation was performed at room temperature using a Gammacell 1000 irradiator (Atomic Energy of Canada. Ottawa, Can ada) under aerobic conditions with a 117Cs source emitting at a fixed dose rate of 13.3 Gy/min, as determined by dosimetry. Subcellular Fractionation. Subcellular fractionation was performed as described (24). HL-60 cells were washed twice with cold PBS and resuspended in 1 ml hypotonie lysis buffer (1 mM EGTA, 1 mM EDTA. 10 mM ß-glycer- ophosphate, 0.5 mM sodium orthovanadate, 2 mM MgCl2, 10 mM KC1, I mM DTT, 40 fj.g/m\ phenylmethylsulfonyl fluoride. 10 /ig/ml leupcptin, and 10 /xg/ml aprotinin, pH 7.2). After swelling on ice for 30-45 min, the cells were disrupted by Dounce homogenization (25 strokes). The homogenate was layered onto 1 ml of 1 M sucrose in lysis buffer and centrifuged at 1600 X g for 15 min to pellet the nuclei. The supernatant above the sucrose cushion was collected and centrifuged at 150,000 X g for 30 min at 4°Cto collect the soluble or cytoplasmic fraction. Purity of the fractions was monitored by immunoblot analysis with anti-PDGF receptor (SC-78, Santa Cruz Biotech nology, Santa Cruz, CA) and antihistone HI (1492-519, Boehringer-Mann- heim) antibodies. Preparation of Cytoplasts. Enucleated cells were prepared as described (25). In brief, a discontinuous density gradient consisting 25, 19, 18, 17. 16, 15, 12.5 and 10% Ficoll was prepared from Ficoll stock solution (50% w/v in water) containing 10 ng/ml cytochalasin B and RPMI 1640. Cells (2 X IO6) were washed twice with PBS, resuspended in 2 ml of 10% Ficoll containing cytochalasin B. loaded onto the gradients, and spun at 25.000 rpm at 30°Cfor 1 h. Cytoplasts recovered from the 15-16% Ficoll interface were washed three times with fresh medium and incubated for l h at 37°Cbefore treatment. 3617 Research. on December 15, 2021. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from