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1748 Humań Molecular Genctics, 1999, Vol. 8. No. 9

celi pellets were then resuspended in ihe isotonic solution at 5 x 10⁶ cells/ml. Aliquots of 250 000 cells were spread out on SuperFrost slides willi a Cytospin at 400 r.p.m. for 5 min. Cells were subseąuently air dried and fixed in cold 70% alcohol for 5 min at 4°C and washed twice for 5 min in cold phosphate-buffered salinę (pH 7.2). For douoie-labelling, slides were then co-incubated for 2 h at room temperaturę with a mouse mono-clonal antibody to RARA (Ab9a(F)(9a-9A6); 1/100] (Prof. P. Chambon. Strasbourg, France) and with rabbit polyclonal IgG STAT5b N-20 or STAT5b C-17 (2 mg/ml; Santa Cruz Bio-technology). After a second incubation with rhodamine-conjugated sheep anti-rabbit antibody (1/40; Oncor) for 30 min at room temperaturę, a finał incubation with FITC-conjugated rabbit anti-mouse antibody (1/40: Dako) for 30 min at room temperaturę was performed. The slides were washed in phos-phate-buffered salinę and mounted with antifade (Oncor).

Southern błotting

Total genomie DNA from lymphocytes or from bonę marrow ceils was extracted with the Nucleon BACC3 DNA isolation kit (Amersham). Southern biot anaiysis was performed follow-ing standard methods. Rearrangemeni of the STAT5b loeus was investisated with the full-Iensth STAT5b cDNA.

w    w-

FISH

FISH anaiysis was performed with PAC 196pl 7 on cyto-genetic preparations from a normal małe. Prometaphase chro-mosomes w^ere prepared foliowing the standard procedures. The D17S258 Smith-Magenis probe (Oncor) w'ere used as a marker for chromosome I7pl 1.2. The D17S258 probe also contains a RARA-specific probe. PAC 2 96p 17 DNA (!7ql2) was labelied by nick-translation (Biotin-Nick Translation Mix: JBoehringer Mannheim) with 16-dUTP-biotin. Suppression of repetitive seouences with Cot-1 DNA follow'ed by co-hybridization of D17S258, RARA and PAC 196p 17 were done following standard methods. Washes, signal detection and amplification were performed according to the chromosome in situ hybridization protocol (Oncor). The chromosomes were counrerstained with DAPI antifade (Oncor; and visualized with a Zeiss Axioplan fluorescent microscopc equippea with a PSI Power Gene FISH System analyser.

ACKNOWLEDGEMENTS

The authors wish to thank Prof. C.C.A. Bernard (Melbourne. Australia), Dr O. Bernard (INSERM U434), Prof. P. Lebiond. F. Wuilque. Drs M.T. Daniel and J. Buisine for helpful discus-sions. They are also indebted to Profs P. Chambon (IGBMC-LGME-U184-ULP) and W.J. Leonard (Bethesda. MD; for the RARA antibody and the STAT5bcDNA, respectively. We are grateful to L. Maury for help with the patiem genomie Jibrary screening and to F. Poły for PAC library screening. We also thank Profs G. Faure and M.C. Bene for help with immuno-cytochemistry and M. Franek and B. Petitjean for technicy! assistance. This work was supported by Association pour la Recherche contrę le Cancer, Ligue Nationale contrę le Cancer. la Fondation contrę ia Leucemie and Association Franęaise de lutte contrę les Myopaihies. C.A. was supported by a fellow-ship from the Ministere de la Recherche et de i* Es pace (no. 94951338083 J.

REFERENCES

1.    dc The. H.. Chomiennc. C.. Lanotte. M.. Dcgos. L. and Dejean. A. (1990) The t(15;17) translocation of acutc promyelocytic leukaemia fuses ihe retinoic acid receptor a gene to a novel transcribed loeus. Naturę. 347. 558-561.

2.    Chen. Z..BrandN.J..Chen. A..Chen. S.J..Tong.J.H.. Wang.Z.Y., W&xmun. S. and Żelem. A. (1993) Fusion between a novel Kruppel-Iike zinc fingcrgene and the rctinoic acid receptor Ot loeus due to a variant t( 11;J7) associated with acute promyelocytic leukaemia. EMBOJ.. 12. 1161-1167.

3.    Redner. R.L.. Rush, E.A.. Faas. S.. Ruden. W.A. and Corey. S.J. (1996) T?ie t(5:17) variant of acutc promyelocytic leukemia expresses a nucleo-phosmin-retinoic acid receptor fusion. Blood. 87. 8S2-8S6.

4.    Wells. R A.. Catzavelos, C. and Kamel-Reid. S. (1997) Fusion of retinoic acid receptor a to NuMA. the nuclear mitotic appararus protein, by a vanant trans-location in acute promyelocytic leukaemia. Naturę Genct. 17. 109-113.

5.    Jonveaux. P.. Le Coniat. M.. Dcne. J., Flexor. M.A.. Daniel. M.T and Berger. R. (1996) Girorr.osomc microdisscction in leukemia: a power tool for the anaiysis of comp!ex chromosomal rearrangements. Cenes Chromosomes Cancer. 15,26-33.

6.    Damell. J.E.Jr i 1997) STATs and gene rcgulation. Science, 277. 1630-1635.

7.    Pcllcgrini. S. and Dusanter-Fourt. I.JI997) The structure. reguiation and function of the Janus kin3ses (JAKs) and the signal transducers and actt-vators of transcription (STATs). Eur. J Biochem., 248. 615-633.

8.    Heim. M.E. (1999) The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J. Recept. Signal Transduct. Rcs.. 19. 75-120.

9.    Wakoa. H.. Gouil!eux. F. and Groner. B. (1994) Mammary gland factor (MGF) is a novc! member of the cytokine regulated transcription factor gene family and confers the prolactin responsc. EMBOJ.. 13. 182-191.

10.    Lin. J.X.. Mietz. J.. Modi. W.S.. John. S. and Leonard. W.J. (1996) Gon-mg of human Stat5b. J Biot. Chem.. 271. {0738-10744.

11.    Merisel. R.. Gouilleux-Gruan. V.. Jahnc. R.. Bcrchtold. S.. Garmann. C.. Liu. X.. Hennighausen. L., Sotiropoulos, A.. Groner. B. and Gouilieux. F.

(1996* Delenon of the carboxy|-termina) transactivation domam of Y1GF-Stań results in sustained DNA binding and a dominant negative pheno-type. Mol. Celi. Biol., 16. 5691-5700.

12.    Gouilleux, F.. Pallard. C.. Dusanter-Fourt. I.. Wakao. H.. Haldosen. L.-A.. Norstedt. G.. Lew. D- and Groner. B. (1995) Prolactin. growth hormone. erythropoietm and granulocyte-macrophaee colony stimulating factor induce MGF*Stat5 DNA binding activity. EMBOJ., 14. 2005-2013.

13.    Welle. T.. Koch. F.. Schuler, G.. Lcchner. J.. Doppler. W', and Heufler. C. (19971 Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT factors in murine dendritic cells. Eur. J. tmmunoi. 27. 2737-2740.

14 Teglund. S.. McKay, C.. Schuctz. E.. van Deurscn. J.M.. Stravopodis. D.. Wang. D.. Brown. M.. Bodner, S.. GrosveId. G. and Ihle. J.N. <1998) Stat5a and Staób proteins h3ve essential and nonessentia!. or redundam. roles in cytokine responses. Celi. 93. 841-850.

15.    al-Shami. A.. Mahanna, W. and Naccache. P.H. (T998) Granulocyte-macrophagc colony-stimulating faclor-actjvated signaling pathways in human oeutrophils. J. Biol. Chem . 273. 1058-1063.

16.    Caldenhoven. E.. van Dijk. T.B.. Tjimensen. A.. Raaijmakers. J.A.M.. Lammers. J.W.J.. Koenderman. L. and de Groot. R.P. <1998) Diffcrential acińation of functionalJy distinct STATS proteins by 11-5 and GM-CSF during eosinophil and neutrophi! differentiation from human CD34-r hemaiopoieiic stem cells. Stern Cells. 16. 397-403.

17.    Welte. T.. Leuenbcrg, D._t Dittel. B.N.. al-Ramadi. B.K.. Xie. B.. Chin. Y.E.. Janeway. C.A.Jr. BothweU, A.L.M., Bottomly. K. and Fu. X.Y. (1999i STATS interaction with the celi receptor complex and stimulation of T-ce!l proliferation Science. 283. 222-225.

18.    Wiiks. A.F. and Oates. A.C. (1996) The JAK/STAT pathwavs. Cancer Sun .27. 139-163.

19.    Mui. A.L.. Wakao. H.. Kinoshita, T.. Kitamura. T. and Miyąjima. A. ()996» Supprc>'ion of inter!cukin-3-induced gene cxprcssion by a C-terminal truncated SlatS: role of Stat5 in proliferation. EM BO J.. 15. 2425-2433.

20.    van der Pląs. D-C.. Smiers, F.. Pouwels. K.. Hocfsioot, L.H.. Lowenberg. B. and Touv\. I.P (1996) Imerleukin-7 in human B celi precursor acute lymphoblastic ieukemia cells and murine BAF3 cdls involves activation of STAT1 and STAT5 mediated via the interlctikin-7 receptor alphacham Leukemia. 10. 1317-1325.

21.    Jmudu. K.. Blorjm.E.T .Nakajima. H.. Horvath-Arcidiacono. J.A . Udy.G.B.. Davc> H.W. ano Leonard. W.J (I99K) StaLSb is essential for natura! killcr ccll-mediated proliferation and cytolytic aciivity.7. Esp Med. 7.2067-2074.