A Somatostatin Analogue Induces Translocation of Ku 86 Autoantigen from the Cytosol to the Nucleus in Colon Tumour Cells

A Somatostatin Analogue Induces Translocation of Ku 86 Autoantigen from the Cytosol to the Nucleus in Colon Tumour Cells
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  Cell. Signal. Vol. 10, No. 4, pp. 277–282, 1998 ISSN 0898-6568/98 $19.00Copyright  󰂩  1998 Elsevier Science Inc. PII S0898-6568(97)00128-9 A Somatostatin Analogue InducesTranslocation of Ku 86 Autoantigen from theCytosol to the Nucleus in Colon Tumour Cells  Jo´ zsef To´ va´ ri, †  Be´ la Szende, †  Jo´ zsef Bocsi, †  Arturo Falaschi, ‡  Andra´ s Simoncsits, ‡ Sa´ ndor Pongor, ‡  Judit E´ rchegyi, §  Attila Steta´ k §  and Gyo¨rgy Ke´ ri §* †First Institute of Pathology and Experimental Cancer Research , Semmelweis University of Medicine ,  U¨llo˜i st.  26,  Budapest , 1085,  Hungary ;‡ International Centre for Genetic Engineering and Biotechnology ,  Area SciencePark ,  Trieste , 34012,  Italy ;  and §Institute of Medical Chemistry ,  Molecular Biologyand Pathobiochemistry ,  Joint Research Organisation of the Hungarian Academy ofScience and Semmelweis University of Medicine ,  Puskin st.  9,  Budapest ,  Hungary ABSTRACT.  Flow cytometric and electron microscopic immunocytochemical studies have been performed inHT-29 human colon tumour cells  in vitro , to determine and localise p86 Ku protein, which is a regulatory subunitof DNA-dependent kinase and a specific binding site for somatostatin. We have demonstrated that HT-29 cellscontain p86 Ku and that the distribution between the cytoplasm and the nucleus is even. After administrationof the somatostatin analogues Sandostatin and TT-232 to HT-29 cells, the p86 Ku content of the cytoplasmiccompartment decreased in the first 4 h. An increase in the content of this protein in the nuclear compartmentwas observed at hour 1 followed by a decrease at hour 4 after treatment. Quantitative differences between thetwo analogues have been observed in this respect. The practical significance of these findings is discussed.  cellsignal  10;4:277–282, 1998.  󰂩  1998 Elsevier Science Inc. KEY WORDS.  Ku 86 protein, Somatostatin, Nuclear receptor, Colon tumour cells INTRODUCTION  effect of somatostatin is mediated by the Ku 86 nuclear pro-tein. Ku 86 has been localised not only in the nucleus, butDNA-dependent protein kinase (DNA-PK) is a nuclear ser-also in the cytoplasm and in the cell membrane, suggestingine/threonine protein kinase that can be activated  in vitro that cellular distribution might determine the cellular func-by DNA fragments, and one of its major cellular targets istion of Ku proteins [5].the anti-oncogen protein p53. It has been suggested that,We have developed a tumour-selective structural deriva-after exposure of mammalian cells to DNA-damagingtive of somatostatin analogues ( d -Phe-Cys-Tyr-D-Trp-Lys-agents, the mitogen-activated protein (MAP) kinase cas-Cys-Thr-NH 2 ; code name: TT-232) that shows strong anti-cade and the DNA-PK are activated simultaneously, whichproliferative and apoptosis-inducing effects but no endocrineresults in the phosphorylation of various transcription fac-effect. We have demonstrated that TT-232 inhibits tyrosinetors and in the stabilisation of p53 through a post-transla-kinases in human colon tumour cell lines and that this inhi-tional mechanism that then activates the G1 checkpointbition correlates with the apoptosis-inducing effect [6, 7].growth-arrest mechanism or programmed cell death or bothThe aim of this study was to demonstrate whether Ku 86[1, 2].is present in HT-29 cells and, if so, what the effect of DNA-PK consists of a 350,000  M r  catalytic subunit thatTT-232 as well as that of Sandostatin—a somatostatin ana-forms a complex with a so-called Ku protein. This Ku pro-logue used in clinical practice—on the expression and sub-tein was discovered as an autoantigen and serves as a regula-cellular localisation of Ku 86.tory or targeting subunit for DNA-PK [3, 4].Ku is actually a family of related proteins and the 86,000- M r  subunit of it is a somatostatin receptor playing a tar- MATERIALS AND METHODS geting role, for example, in activating RNA polymerase by Tumour Cells and Culture Conditions DNA-dependent kinase and in regulating protein phospha-tase-2A. It has been suggested that the anti-proliferativeHT-29 human colon carcinoma cells were maintained  invitro  as monolayer cultures with the use of six-well Greiner *Address all correspondence to Gyo¨rgy Ke´ri, P. O. Box 260, Budapest, dishes (Kremsmu¨nster, Austria) in RPMI 1640 medium sup- Hungary-1444. Tel: 36-1-2662755/ext. 4068 or 4094; Fax: 36-1-266480.Received 10 June 1997; and accepted 18 August 1997.  plemented with 10% foetal bovine serum (FBS) (ICN-  278  J. To´va´ri  et al.  FIGURE 1.  P86 Ku protein con-tent in HT-29 carcinoma cellsby flow cytometry. Values arethe mean   SEM;  P  is not sig-nificant. Determination of Ku 86 Content Flow, Irvine, Scotland) at 3  C in a humidified atmosphereof 5% CO 2  and 95% air.The HT-29 colon tumour cells were detached from theHT-29 cells were incubated  in vitro  with somatostatin an-wells with 0.02% EDTA and were fixed in absolute metha-alogues TT-232 and Sandostatin (Sandoz Pharma LTD,nol for 10 min; the non-specific binding sites were blockedSwitzerland) for 1 or 4 h at a concentration of 10  g/mL.with FBS [1  3 in phosphate-buffered saline (PBS)] for 30min. The fixed cells were incubated with the primary Ku 86antibody (1  100 in PBS) for 60 min, with biotinylated anti- In Vitro  Anti-Proliferative Assays rabbit immunoglobulin G (Amersham, Little Chalfont,Approximately 350,000 cells were cultured in 2 mL of  UK) (1  200 in PBS) for 60 min, and with streptavidin fluo-RPMI 1640 culture medium in a well and incubated for 24 rescein (Amersham) (1  200 in PBS) for 30 min and thenh with 1 and 10  g/mL doses of TT-232 and Sandostatin in analysed by flow cytometry (FACStar, Becton Dickinson,the absence of FBS. After trysinisation and dispersion, the Mountain View, CA). Cells were considered positive forcells were dyed with trypan blue and counted in a hemocy- the presence of Ku 86 when the fluorescence intensity wastometer. above that of 90% of the control cells (prepared by omittingthe incubation with primary antibody). Preparation of AntibodiesTransmission Electron The antibodies were raised in rabbit against the recombi-  Microscopic Immunocytochemistry nant 86,000  M r  protein, which was prepared as describedpreviously [8]. For the first injection, the purified 86,000  M r  The detached (with 0.02% EDTA) and glutaraldehyde-subunit of the Ku antigen was mixed with Freund’s com- fixed tumour cells were embedded in Spurr’s low-viscosityplete adjuvant (Sigma Chemical Co., Budapest, Hungary). embedding medium (Polyscience, Warrington, PA). ForThe rabbits were then boosted at least four times every 2 electron microscopic immunocytochemistry, sections wereweeks prior to the final bleeding. The antibodies were puri- cut with diamond knives and mounted on gold grids; poly-fied by using protein A-Sepharose (Pharmacia, Uppsala, clonal Ku 86 antibody was used. The sections were blockedSweden) as described in the literature, and the specificity of  in normal goat serum (1  5 in PBS) and incubated with thethe serum was checked by Western blotting on homoge- primary antibody at room temperature for 1 h, and with thenates of HT29 cells as described previously [8] and found secondary gold-labelled anti-rabbit antibody (AuroProbeidentical with that prepared against the Ku 86 subunit puri- EM GAR G10 IgG, Amersham) for 1 h. Negative controlswere incubated with the gold-conjugated secondary anti-fied from HeLa cells [9].  Translocation of Ku 86 Autoantigen in Colon Tumour Cells  279  FIGURE 2.  Quantitative changein gold particles representingp86 Ku proteinin thecytoplasmofHT-29cellsafterSandostatinand TT-232 treatment. Valuesare the mean  SEM;  P  0.05  vs.  control. body only. Sections were viewed, without contrasting and treatment. Figure 5 shows immunogold staining of Sando-after staining with uranyl acetate, in a CM 10 electron mi- statin-treated cells at hour 4, indicating a decrease in cyto-croscope (Philips, The Netherlands). plasmic gold particles. However, a slight non-significantincrease in the number of gold particles in the nucleus wasobserved after TT-232, and a significant increase of the Statistical Analysis same occurred after Sandostatin treatment at hour 1 (Fig.The results of   in vitro  studies were evaluated by the Mann-3). Figure 6 shows immunogold staining of Sandostatin-Whitney U-test, and Student’s  t -test. Flow cytometric datatreated cells at hour 1, with dense labelling of the nucleus.were evaluated by the Consort 30 system (Becton Dick-At hour 4, the TT-232-treated samples showed a slight de-inson).crease in the number of gold particles, whereas the Sando-statin-treated samples revealed a marked decrease far belowthe control value. RESULTS The effects of TT-232 and Sandostatin on the prolifera-The presence of p86 Ku protein in HT-29 cells was demon-tion of HT-29 tumour cells were compared. In a representa-strated by both flow cytometry and electron microscopictive experiment, TT-232 at a 10  g/mL dose showed a 59  immunocytochemistry. Figure 1 shows the results obtained6% decrease in the number of tumour cells, but Sandostatinby flow cytometry. A non-significant increase of the fluores-resulted in only a 21  12% decrease. At a 1  g/mL dose,cence intensity was measured at hour 1 after TT-232 treat-both peptides were less effective and the difference in thement followed by a similarly non-significant decrease. San-antiproliferative effect of the two somatostatin analoguesdostatin caused a non-significant drop in the amount of p86was less striking (Fig. 7).Ku protein both at 1 h at 4 h after treatment. In regard tothe results obtained by the immunogold method, the grainnumber representing p86 Ku protein was nearly equal in the DISCUSSION cytoplasm and in the nucleus (Figs. 2 and 3). Figure 4 showsIn our earlier work, we studied the signalling pathways of immunogold staining of a control cell, with equal distribu-HT-29 human colon tumour cells [10, 11]. Recently, wetion of gold particles in the nucleus and cytoplasm. Thedemonstrated that our novel somatostatin analogue (TT232),number of gold particles in the cytoplasm decreased signifi-which has strong  in vitro  and  in vivo  antitumour activity, in-cantly by the administration of both TT-232 and Sando-statin (Fig. 2) at hour 1 and decreased further at hour 4 after duces a strong apoptotic effect in HT 29 human colon tu-  280  J. To´va´ri  et al.  FIGURE 3.  Quantitative changein gold particles representingp86 Ku protein in the nuclei ofHT-29 cells after Sandostatinand TT-232 treatment. Valuesare the mean  SEM;  P  0.05  vs.  control. mour cells [7]. In the present work, we have investigated could be demonstrated in HT-29 cells by flow cytometryand electron microscopic immunocytochemistry. Moreover,whether the previously described nuclear somatostatin re-ceptor—p86 Ku, which was suggested to play a regulatory both somatostatin analogues applied by us influenced thequantity and especially the distribution of p86 Ku moleculesrole in p53-mediated apoptosis—is present in the HT29cells and whether it mediates the apoptosis-inducing effect between the cytoplasm and the nucleus, detected by immu-nogold staining method. The changes were characterised byof somatostatin analogues. In accordance with the findingof Le Romancer  et al.  [5], the presence of p86 Ku protein a decrease in the number of the gold particles in the cyto-  FIGURE 4.  Untreated HT-29cells. Gold particles representp86 Ku protein, which is evenlydistributed in both the cytoplasmand the nucleus.  Translocation of Ku 86 Autoantigen in Colon Tumour Cells  281  FIGURE 5.  HT-29 cells treatedfor 4 h with Sandostatin, whichcaused a decrease in cytoplasmicgold particles representing p86Ku. plasmic compartment and by an initial increase and a con- cal importance of p86 Ku in the mediation of the action of somatostatin. The observed differences in the degree of secutive decrease in the number of gold particles in the nu-clear compartment. Quantitative differences were notable down-regulation of this intracellular binding site of somato-statin caused by the 4-h treatment with the various ana-between the two analogues, but the tendency was similar.Our previous studies showed that binding sites for lutein- logues may have to be considered when these analogues areadministered for therapeutic reasons. Binding of radiola-izing hormone–releasing hormone (LHRH) also are presentboth in the nucleus and in the cytoplasm [12], and these belled somatostatin analogue TT-232 to HT-29 cells is nowunder investigation and may provide more data on theLHRH receptors were concentrated in the nucleus after D -Trp-6-LHRH administration [13]. Binding of radiolabelled mechanism of action of this peptide, the end result of whichis the induction of apoptosis of treated cells [15].LHRH analogue to the cell surface and to the nuclear chro-matin also was shown [14]. According to our present stud- Our data suggest that Sandostatin might exert its anti-proliferative effect through Ku 86—for example, by targetingies, the distribution of p86 Ku between the nucleus and cy-toplasm is even. The changes in the distribution upon DNA-PK to p53, as suggested for native somatostatin byRomancer  et al.  [5], whereas TT-232 probably does not ex-somatostatin-analogue treatment indicate the real biologi-  FIGURE 6.  Sandostatin-treated(1 h) HT-29 cells; p86 Ku pro-tein is concentrated in the nu-cleus.
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