rheb, Gene Responsible for UV Susceptibility of Human Cells

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1 heb, Gene Responsible fo UV Susceptibility of Human Cells K. Kita 1, K. Kikuno 2, Y. P. Wu 1,.J. Hiano 3, H. Yamamoi 3, and N. Suzuki 1 Depatment of 1 Biochemisty, 2 Laboatoy Medicine, 3 Sugey, Chiba Univesity School of Medicine, Inohana 1-8-1, Chuo-ku, Chiba city, Chiba 260, Japan INTRODUCTION Iadiation of human cells with fa-ultaviolet light (UV, pincipally 254-nm wavelength) induces vaious cellula events such as DNA damage, cell death and mutation (1, 2), via induced expession of seveal genes (3, 4). Some of the genes may play oles in the pleiotopic events of UV (1, 4). Cell lines with discepancy of UV susceptibility seem to be valuable fo identification of such UV-induced genes (5). We theefoe attempted to seach fo genes involved in the susceptibility of cells to UV effects, compaing alteation of gene expession levels afte UV iadiation between UV-sensitive RSa cell line and UV-esistant cell line, UV -1 cells. UV -1 cells wee deived fom RSa cells and showed inceased esistance to UV-induced cellkilling. Levels of thei expessed genes wee analyzed by a PCR-based mrna diffeential display method, following confimation by Nothen blot analysis. We hee epot one gene esponsible fo the UV susceptibility of human cells. MATERIALS AND METHODS Cells and cultue conditions. RSa and UV -1 wee descibed peviously (6, 7). Cells wee seeded in 100-mm dish, gown until subconfluent, and then iadiated with 10 J/m 2 UV (8) o mock-iadiated. Fou h afte UV iadiation, total cellula RNA was isolated fom the cells as peviously descibed (8). PCR-based mrna diffeential display and Nothen analysis. Diffeential display was pefomed as descibed (8, 9), by using RNAmap kit (GenHunte, Bookly, MA, USA). cdnas cloned afte the diffeential display method wee used as templates fo sequencing as descibed (8). Alteation of the mrna expession levels fo the cloned cdnas was confimed by Nothen blot analysis. Antisense study. Fo antisense study, we used highly puified phosphothioate antisense oligonucleotides which was diected against the amino-teminal egion specific fo human Ras homolog eniched in bain (Rheb) mrna (antisense-oligo) o consisting of andomized sequence showing the same nucleotide composition (andomoligo) (Geine Japan, Tokyo, Japan). Cells wee cultued with EMEM containing 10% calf seum and the oligonucleotides fo 5 days afte seeding. Duing the cultue peiod, the medium was efeshed evey 2 days. The cells wee utilized fo examination of suvival activity afte UV iadiation by colony fomation assay (8, 10). RESULTS RT-PCR was pefomed using 20 diffeent combinations of andom pimes. Thiteen cdna bands, which had moe than two-fold o less than a half intensity of that of mock-iadiated cells, wee selected (data not shown). One up-egulated clone among the cdnas was highly homologous to Ras-elated GTP-binding potein, human Ras homolog eniched in at bain (Rheb) (11, 12). The mrna expession of Rheb was futhe examined by Nothen blot analysis. In UV-iadiated RSa and UV -1cells, the expession levels of Rheb mrna wee found to be inceased o deceased to only a little extent, when compaed with those in mockiadiated cells (Table 1). Table 1. UV induction of Rheb mrna expession levels Cells RSa UV -1 UV induction a) a) The Rheb mrna expession levels 4h afte UV iadiation wee analyzed by Nothen blotting and divided by GAPDH mrna levels. The levels wee shown as the expession elative to those afte mock iadiation. 1

2 Howeve, Rheb mrna expession levels wee found to be moe abundant in UV -1 than in RSa cells in conditions without iadiation (Table 2). When functional analysis using antisense oligonucleotides fo Rheb was Table 2. Expession levels of Rheb mrna in logaithmically gowing cells Cells RSa UV -1 Relative levels a) a) The Rheb mrna expession levels wee divided by GAPDH mrna levels. The expession levels of RSa cells was designated as 1.0, and the expession levels of UV -1 cells ae shown as elative levels to those of RSa cells. pefomed, antisense-oligo-teated UV -1 cells showed moe inceased esistance to UV cell-killing than did unteated cells (Figue 1). Random-oligo did not change UV suvival of both the cells (data not shown). 100 antisense-oligoteated-uv -1 UV UV (J/m 2 ) Figue 1. Faction of colony fomes plotted against vaious doses of UV. Antisense oligonucleotides teatment and colony fomation assay wee pefomed as descibed in "MATERIALS AND METHODS". Bas indicate the standad deviations. 15 2

3 DISCUSSION The pecise molecula mechanisms undeling UV sensitivity of RSa and UV -1 cells emain unclaified. In the pesent study, we seached fo genes, expession levels of which alteed afte UV iadiation, using mrna diffeential display and Nothen analysis. Expession levels of human Rheb mrna wee inceased in UVesistant UV -1 cells to a geat extent. Futhemoe, ou esults, showing that UV -1 cells teated with antisense oligonucleotides fo Rheb mrna became moe esistant to UV cell-killing than unteated cells, suggest that Rheb is involved in susceptibility of UV -1 cells to UV cell-killing. To ou knowledge this is the fist epot that Rheb is involved in the susceptibility of human cells to UV cell-killing. Rheb was a ecently identified as a membe of the Ras supefamily (11, 12, 13). UV esponse in mammalian cells is mediated by a signal pathway involving Ras (2, 14). Rheb may be involved in cell polifeation inhibition and/o cell-killing afte UV iadiation, and may play potective oles against UV damage in UV -1 cells. By contast, Rheb might not adequately function in RSa cells. ACKNOWLEDGMRNTS This wok was suppoted in pat by a gant-in-aid fom the Smoking Reseach Foundation, Mishima Kaiun Memoial Foundation fo eseach found, Gound Reseach fo Space Utilization pomoted by NASDA and Japan Space Foum, the Sumitomo Foundation, the Japan Atomic Enegy Reseach Institute, by the contact on the Nuclea Safety Reseach Association, and gant-in aid fom the Ministy of Education, Science and Cultue, Japan. REFERENCES 1. P. Helich, H. Ponta and H.J. Rahmsdof, Rev. Physiol. Biochem. Phamacol. 119, (1992). 2. R.M. Tyell, BioEssays 18, (1996). 3. S.M. Keyse, Sem. Cance Biol. 4, (1993). 4. A.J. Fonace, Annu. Rev. Genet. 26, (1992). 5. C.A. Bill and P.J. Tofilon, Int. J. Radiat. Biol. 65, (1994). 6. N. Suzuki and A. Fuse, Mutation Res. 84, (1981). 7. N. Suzuki, Mutation Res. 125, (1984). 8. Y. Higuchi, K. Kita, H. Nakanishi, X-L. Wang, S. Sugaya, H. Tanzawa, H. Yamamoi, K. Sugita. A. Yamaua and N. Suzuki, Biochem. Biophys. Res. Commun. 248, (1998). 9. P. Liang and A.B. Padee, Science 257, (1992). 10. K. Kikuno, K. Kita, J. Nomua, T. Hiwasa, H. Yonemitsu and N. Suzuki, Biochem. Biophys. Res. Commun. 253, (1998). 11. P.S. Gomov, P. Madsen, N. Tomeup and J.E. Celis, FEBS Lettes 377, (1995). 12. N. Mizuki, M. Kimua, S. Ohno, S. Miyata, M. Sato, H. Ando, M. Ishihaa, K. Goto, S. Watanabe, M. Yamazaki, A. Ono, S. Taguchi, K. Okamua, M. Nogami, H. Taguchi, A. Ando and H. Inoko, Genomics 34, (1996). 13. K.Yamagata, L.K. Sandes, W.E. Kaufmann, W. Yee, C.A. Banes, D. Nathans and P.F. Woley, J. Biol. Chem. 269, (1994). 14. D. Engelbeg, C. Klein, H. Matinetto, K. Stuhl and M. Kain, Cell 77, (1994). It was epoted that Rheb was synegistic with Raf-1 in tansfoming gowth popeties of NIH 3T3 fiboblasts and also induced neuite outgowth potentiated by NGF and camp in PC12 cell (14). We peviously epoted that inceased expession levels of nucleophosmin (6) and syndecan-1 (7) genes esult in the esistance of cells to UV cell-killing. 3

4 Howeve, Rheb was also suggested to antagonize Ras function by foming inactive complexes with Raf-1 in NIH 3T3 cells (15). 5. (14) C. F. Rosen, R. Poon, D. J. Ducke, Am. J. Physiol. 268, C846-C855. (15) H. F. Abts, K. Beuhahn, G. Michel, K. Kohe, P. Esse, T. Ruzicka, Photochem. Photobiol. 66 (1997) (16) T. M. M. Vogt, J. Welsh, W. Stolz, F. Kullmann, B. Jung, M. Landthale, M. McClelland, Cance Res. 57 (1997) (18) N. Suzuki, T. Kuwata, Mutation Res. 60 (1979) (19) P. Chomczynski, N. Sacchi, Anal. Biochem. 162 (1987) (20) T. Ishizuka, K. Kita, T. Sonoda, S. Ishijima, K. Sawa, N. Suzuki, M. Tatibana, Biochim. Biophys. Acta 1306 (1996) (21) M. Enst, U. Novak, S. E. Nicholson, J. E. Layton, A. R. Dunn, J. Biol. Chem.274 (1999) (22) N. Suzuki, J. Nishimaka, T. Kuwata, Mutation Res. 106 (1982) (25) E. C. Fiedbeg, DNA Repai (1985) pp , W. H. Feeman and Company, New Yok. (26) G. C. Walke, Cellula and Molecula Biology (ed. F. C. Niedhad, J. L. Ingaham, K. B. Low, B. Magasanik, M. Shaechte and H. U. Umbage) (1987) pp , Ameican Society of Micobiology, Washington DC. (27) P. Angel, H. J. Rahmsdof, A. Poting, P. Helich, Cance Cells 3 (1985) (28) M. Gamyn, M. Yaa, N. Holbook, B. A. Gilchest, J. Lab. Invest. 65 (1991) (29) C. Campbell, A. G. Quinn, B. Angus, P. M. Fa, J. L. Rees, Cance Res. 53 (1993) (30) B. Stein, H. J. Rahmsdof, A. Steffen, M. Litfin, P.Helich, Mol. Cell. Biol. 9 (1989) (31) G. Hemann, M. Wlaschek, T. S. Lange, K. Penzel, G. Goez, K. Schaffette-Kochanek, Exp. Dematol. 2 (1993) (32) N. Suzuki, H. Suzuki, T. Kojima, K. Sugita, Y. Takakubo, S. Okamoto, Mutation Res. 198 (1988) (33) N. Suzuki, H. Suzuki, Mutation Res. 202 (1988) (34) K. Sugita, N. suzuki, H. Niimi, Mutation Res. 357 (1996) (35) K. Sugita, N. Suzuki, Y. Higuchi, K. Kita, Y. suzuki, A. Lehmann, Mutation Res. 408 (1998) (40) M. A. Guthidge, M. Seldin, C. Basilico, Oncogene 12 (1996)

5 Figue legends Fig. 1 Diffeentially displayed cdna bands in RSa cells with (10 J/m2) and without UV iadiation. Fou h afte UV iadiation, total RNA was extacted and subjected to diffeential display analysis, as descibed in Mateials and methods. The aow indicates the diffeentially displayed cdna band. Fig. 2 Nothen blot analysis of RSa cells with (10 J/m2) and without UV iadiation using as pobes 4 diffeentially expessed cdna fagments. Fou h afte UV iadiation, total RNA was extacted and 12 mg of DNase-teated total RNA was applied to each lane. The 4 diffeentially displayed cdna fagments wee used as pobes and hybidized as descibed in Mateials and methods. Appoximate mrna sizes ae shown in kb. Fig. 3 Alteations of mrna expessions fo 4 clones afte UV iadiation in vaious cells. RNAs wee extacted 1, 4 and 12 h afte UV iadiation (10 J/m2) fom RSa, UV-1 and FF cells, and analyzed by Nothen analysis as descibed in Mateials and methods (A). The mrna level was quantified by the imaging analyze, nomalized fo the value of GAPDH mrna as descibed in Mateials and methods, and given as a pecentage of contol without UV iadiation (B)., RSa cells;, UV-1 cells;, FF cells. Fig. 4 (A) Effects of AS-oligo nucleotides teatment on Rheb mrna expession in RSa and UV-1 cells. Expession levels of Rheb mrna in contol (lanes 1 and 5), the andom-oligo-teated (lanes 2 and 6), and the AS-oligo-teated (lanes 3 and 7) cells, wee assessed by RT-PCR, as descibed in メ Mateials and methods モ. The AS-oligo-teated cells wee iadiated with UV and 1 h afte UV, Rheb mrna expession levels wee also analysed (lanes 4 and 8). Rheb mrna expession levels wee nomalized to GAPDH mrna levels, and epesented as elative atios to those of contol cells. (B) Faction of colony fomes plotted against doses of UV in contol ( ), the andom-oligo-teated ( ), and the AS-oligo-teated ( ) cells. Bas indicate the standad deviations. iadiationthe human homolog of FIN14 mrna expession was makedly inceased afte UV iadiation in RSa cells, but was not o less inceased in UV-1 cells and human fetal fiboblast cells. One was down-egulated afte UV iadiation and then ecoveed in RSa cells, with alteation essentially simila to that in the othe 2 cell lines. These genes might also be elated to UV sensitivity in the cell lines. Thus, it seems likely that the diffeential display method using cell lines with discepant UV sensitivity is useful fo identification of genes involved in UV esponse. and, while those of two mrnas fo unknown genes wee down-egulated ae highly sensitive to cell-killing and mutagenic effects of iadiation with fa-ultaviolet light (UV, pincipally 254-nm wavelength), Although we could not pecisely evaluate the alteation of the mrna levels fo NUD103-2 afte UV iadiation because of weak and boad signals on Nothen analysis, the levels in RSa and FF cells tended to be deceased 1 h afte UV iadiation and the low levels continued up to 12 h, and those in UV-1 cells appeaed to be slowly deceased up to 4 h and ecoveed 12 h afte iadiation (data not shown). 5