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Varda Rot Professor Phene Fax + 972 E-Mail COVER Enclose~ The Cm 1. 2. 3. o ,125 @ WEIZlvlAN.~ .~ase find a grant application for: For Tobacco Research USA Inc. a Rotter Ph.D. p53-dependent apoptosis and cell differentiation; shared ess to be reached: Phone + 972 8 343225, 2722 Fax + 972 8 344125 E-Mail LCROTTER @ WICCMAIL.WEIZMA_N-N.AC.IL ~tion of the project: 3 years direct cost: US$ 85,000

$ ar~l~ R0tter~ ?mfessor Phone + 972 g ~:a× ÷ 972 8 34 The Counci 900 Third A New York, U.S.A. Dear Sirs, Please find dependent pathways'.' 43225,272~ 125 . for Tob:~tc~:c:, ,venue q.Y. 1022 enclosed my P.relimina: . apoptosis and cell d " by Va:~ da ]~otl,er. Yours si Varda 1~ Profess~ 8 August 1995 :y Application entitled "p53- ifferentiation; shared molecular ncerely, otter, Ph.D. r

stability c,f 'r::e ~r;n~] ~enome ~ ] 9.-94), Over,~xj-ression ,: f ~5) m:~v #~rce ~he c ell ~c ex9 f~m :ell cycle and obse~aticn ~ha~ exiO'c~,sic,,n cf wiifi-bpe p55 i~ the nmim' bi] ;'.'>eioid celt line, induced eel; apc.p~osis. manifested by ~peciflc cc'~ :r:~o~dJm]c,~>' and ~ypica? DNA fraRmentation (25, 26), ~fis wa~ fl~tlher by data showing lhm ind~:~c~ion cl wild-lype p53 in n hum, an colon ~n~or deriveg ¢:e~] ]iz~e (EB) led ~o apoptosis The conclusion tha~ p53 it, ~ssociated with celt diffen~nfi;~fion was mostly derived trom the obse~'ation tha Ir~sf~lio~ ef wild type E53 ime cells did not cause an irnmediale cell gro~h a~esb but rmEer lmm~tted the ~cu~ence of celhflar events Jml were associated wiO~ lhe mmfifeslation of diffcrenlialion parameters. In lhe c~ of the B-eel! differe~fia~ion pa%way, we found 1ha* imroducfion of wild b'pe p53 expression at rather advanced fl~e cells into a mo,e differentiated phenotylX: (]5~ 29, 30). ]n nom~a~ bone marrow cells, it was suggested ~hat w~ld type p53 promin may play a role in hematopoetic cell maturation, possibly by contributing the inhibition of prolifmation flint occurs during temfinat differentiation (33). In leukemic cell lines such Friend vires t~m~siom~ed ewthroleukemia marine cells, carrying a lemperalure-sensitive ('~s) p53 construct, an a~t in the G0/G 1 phase of flm cell was observed in ll~e presence of tim wild type confonnafion. ~fis was associated witt~ a rapid loss in cell viability ~d induction of hemoglobin synthesis (34), In K-562 an ewthroleukemia truman cell line, reconstimtion of constitutive expression of wild ty~ p53 induced hemoglob~ synth~is (35). In m,oflmr human hemalopoiefic cell Ene, HL. 60 derived from a promyel~ytic leuke~a, expression of wild type p53 promm by a Ts p53 eDNA, induced differentiation of the cells through g~ulocytic pathway (36). Wild type p53 was also found to induce differentialion of norm~ keratinocytes ~d epithelial ce]~s nmnifes~cd by an b~c~e~e b~ the pro~ucti0n of squamons differentiation nm~kers such as involuc (37, 38). 2. Goals and Spedfic Aims Wtm obse~ ration that wild type p53:. on the one hm~d: caw induce cells ~o ~mdergo apoptosis and, on the hand, leads ceEl~, towards differentiation mi~,e'~; the questiox., oi-whethe~ these two p53~ dq~endent events share similar pathwa5~. Can boff~ flwse processe.~ ecc:m concon-fitm~tlv in a Eiven cell, ox is il that so.me cell select the apop~o,Ac pathway, whereas ofl~e,r~- tamer similax pSB-{ndueed conditions will undergo cell differentiation. 'Jhe po~,sibi]i~y also exis,~, 'aha~ in flw same given cell p5B can induce eithe~ apoptosis or cell different iation. is rapid ~md gi",c> ~is~ r, c>:cc- :, Vo~eh~ ]e".,e:]:~ bnEh~' to :~ cl,va~i:.,. On ~Le o~t~e~ haml:. ~hc ~aw, criptional corm ~he

human EL-O). a ?r.c-mvelocvtic ~53 follow changes !n 'A~eir'biolo~iea] acti expressed undo: ;. w~mai physiologica/ r,'pe p53 protein ~a) induce HL-60 l~rotein induced ccjs Io undergo an a¢ cells to undergo c,~ll differentiation at cell to choose between these two p53 cells ~t a given time point (fo~ more 4. Experimental Design and Prt To investigate the molecular m growth arrest, apoptosis and different approach will consist of the introduct p53Val135 plas~id (41). At 37.5°C, whereas at 32°C, the expression of th experiments is to induce in the same ~ and evaluate the expression of specifi of the expression of the specific gene prepared according to published sequ In order to be able to identify n or cell differentiation and to permit tl might be induced or suppressed folio' Transcriptase-PCR (DDRT-PCR) (4~ L12 early pre B-cells: In our earlier studies we found malignant and instead of developing : suggested that mutant p53 functions 1 transferred with constitutive p53 exp: levels of wild type p53 (15, 29). No the same conditions we generated lar ~rotein. The question that we would li~ expression of high levels of wild typ~ (41). Cells will be infected with pL> selection. In our preliminary experin individual subclones will be screener PAb-242 monoclonal antibodies. O~ expression of wild type p53 and gro~ induce cells ~o undergo apoptosis wh following shifting the cells to 32.5°C M1 myeloM cell lines The M1 p53 non-producer cell was found that induction of wild typ~ the Go/G1 or at the S phase (25, 26). In our preliminary studies we I derived virus (41) and obtained a ser generated high producer cell lines an manuscEpt no 2). We noticed that i~ which allows tee expression of wild apoptosis. However, cell lines whic cycle and no apoptosis was evident. cell differentiation. tchanism ~ha: underlie', the K,.nction of v, ild w>e p55 h: ~he h~dn:f.on of cd! mion ~e wiE use a varimv of ce]l b, po w~:kh ]"ack p53 e~pression. O~r on of an inducible p53 e~,pression such as dm ~emW~a~are sensifivo ('F<~ the e~pre~sed p53 is e~:pected 1o exhibit tim m~mn~ pdS p~c.~ein cc~n%n~mfi~,n ~ wild tYt~ p53 pw~ein c<;fi,rmation is e:tpec~ed. The ~dera oi ~hesc :ell lines a p53-dependcm apeptosis or a p53- dependem cell differentiation z genes ..... which are knrwn "to take pa~ in F..~2"cte-penden~ ..... pa~hw~ yd. ~.. ..... Anatv<~.,.. 3~CeS, ew genes thin might be down regulateA following p53-dependem apoptosis e comprehensive comparison between these gene families and Jao.sp 0mr ~ing mutanl p53 expression, we will adapt Hm Differential Display Rever~e ). The following experimental systems will be nsed: that introduction of a mutant p53 into L12 cells cause them to become more egressed tumors, they developed lethal tumors. These results slrongly ~y a "gain of function" mechanism (43). When ttie L I2 cells were :ession vectors, we generated a number ~f clones, all of which expressed low ?53 high producers were, obtained (15). It should be emphasized tha~ unde~ ,~e numbers of mutant p53 derived clones which expressed high bvels of to examine is whether L12 cells could also undergo apoptosis lollowing p53 protein. To that end we will use the p53 Ts ~enovi~al expression vector iSNp53Val135 derived vin~s and pools will be snbjected Io neomw:m dnL~' ~ents we already managed to generate pools ot iMected L12 cel ls~ qhe by Western analysis and FACS analysis rising the anti p53 PAb-421 and ce single cell clones are isolated they will be shifted into 32°C to allow ,th parameters will be evalnated. We expecl that the p53 high producexs wiil ~reas Ihe i)53 tow proflncer cell lines will ~mde~go cell differen:iafio× Cell differentiation will be measured. line was d~orouglfly ir, vesd~,.ated with respect to p:'B indn~:ed at<3~iosi~. t)515 e:~p~ession induced cells to undergo ul:oplo~.i:, c:ithe~ when ave infected the M 1 p53 non-producer celt lin.: ies of deriw:d cell lirms ~hich express var]u~dc .duction of wild tF'gm p55 e:~pression:, foil< .xin£ :yl~ p53 imh:~ced ceEs exFressing high lever h exp~e.~s lo~x levels ol p5?, Frciein showed ,>]> Oargo,fl i~ t:, ex~nine l~e possibility of <he-h .~

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VARDA ROTrER Ph.D. ~t of Cell Biology The Weizmana/mtimte of Science Born 13 June, 1947, in Ansbach, Germany Inunigrated in 1948. Citizenship: Israeli. Mm'ried, two daughters B,Sc. (cure laude), 1969, in Microbiology and Biochemistry, Bar-Ilan univ, Ramat-Gan, Israel M.Se. (cure laude), 1971, in Cell Biology Department of Life Science, Bar-Ban Univ. Ramat-Gan, Israel. Ph.D, 1976, Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel. POSITIONS: 1993 1992 1985-1992 1989-1990 1981-1985 1979-981 1976-1979 Chairperson of the Depamnent of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel Professor, Dep. of Cell Biology, The Weizmann Institute of Science, Re~ovot, Israel Associate Professor, Dep. of Cell Biology, The We're'harm Institute of Science, Rehovot, Israel Visiting Professor on sabbatical leave, c/o Prof. Arnold Levine, Dep. of Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey USA. Senior Scientist, Department of Cell Biology, Weizmann Institute of Science,Rehovot, Israel. Research Topic: Molecular mechanisms controlling the expression of the p53 tumor antigen. Postdoctoral fellow,Center for Cancer Research, Massachusetts Institute of Technology, Carnbddge, MA, USA. Supervisor: Prof. David Baltimore. Research Topic: p53 expression in retroviral transformed cells. Scientist, postdoctoral fellow, Department of Cell Biology, The Weizmann Institute of Science. AWARDS: 1979 1982 1982 1983 1986 1986 1987 1989 1993 1993 1994 Public Health Service International Research post-doctoral Fellowship Israel Cancer Research Fund-Fellowship Incumbent of the Norman and Helen Asher Chair for Cancer Research Special Fellow, Leukemia Society of America Incumbent of the Norman and Helen Asher Professorial Chair for Cancer Research M. Levinson Award for Molecular Biology, The Weizmann Institute Career Development Award from the Israel Cancer Research Fund UICC-Eleanor Roosevelt International Cancer Research Fellowship Israeli Woman of Distinction Award of the Woman's League for Conservative Judaism Feher Prize in medicine Namat award ." EDITORIAL BOARDS: Member, Editorial Board of Oncogene, 1986-date Member, Editorial Board of International Journal of Cancer;Predictive Oneology. 1994-date LIST OF PUBLICATIONS 26. Rotter, V., Witte, O.N., Coffman, R. and Baltimore, D. (1980). Abelson-murine leukemia vires- induced tumors elicit antibodies against a host cell protein, pS0. J. Virol. 36:547-555. 27. Rotter, V., Boss, M.A. and Baltimore, D. (1981). Inct~med concentration of an apparently identical c~llular protein in cells transformed by either Abelson murine-leuk~mia virus of othear transforming agents. J. Virol. 28:336-346. 28. Rotter, V. (1983). p53, a transfornmtion-r~lated e~llq.lar encoded protein, can be.used as a biochemical marl~r for tl~ det~tion of primary minor cells. Proe. Nati. Aead. SCi. 80:2613.-2617. 59. it.error, v. ann ~_otamer, m. tt~'o?, p~ aria lmnnn maligaaaeies. Adv. Cancer Re~., 57:257-271.

61. Shaul~y, G., Ben-Ze'ev, ~_ and Rotter, V. (1990). Subcellular di~m'betion of the p53 prou:in dming-.~ 62. Shsnlsky, G., C_mldfmger, N., Ben-Ze'cv, A. and Ro~er, V. (1990). Nuclear accumulation of p53 Ceil Biol. 1~. 63. Vogeh~i~, B. (1991). 64. P,~e~,D, Rot~V.,,~d~D. (199D. ~~~el~3pn~,o~i~med'~sed - Sh~ky, S., Goldfi~er, N., Peled ~11 diffe~ent~tion i~ vitro. Proc. N~. Acid of Sci. 88:898~-8986 6~. Shaul~ky, G., Goldfing~r N., ,nd Rotter ¥., 0991). Al~r~tion in Tumor D~velopment ~ ~[vo 67. Sho~t-~ O., B~h~ P., ~ich Z., ~d Ro~, V., (I~1). A DNA bi~g do~ ~ ~nmin~ in ~¢ C~nus of wiM ~pe p53 protein. Nucleic Aci~ R~ 19: 5191-5198. 68. Sh~Is~, G., Gold~r, H., TosS, M.S., ~v~e, ~. ~d R~ter, V. (1~1). Nucle~ l~i~tion is ~sen~al for ~e ~fiviW of p53 pin,in. Oncogene 6: ~55-~5. 72. ~nd~sm~, Y., P~o, M., Dm~ G., Roar, V, Fus~nig, N., ~d ~mchi, A., (1992). M~cafions of cell ~clv con~olHng nucle~ proteus by T~-b in HaCat k~r~y~ cell line. Oncogvne 7, 1661-1665. 73. Ronen, D., Tei~ Y. Gol~n~r, N., ~d RotOr V. (19~). Exp~ssion of Wild-~pv ~d Mu~t p53 ~oteins by R~ombin~t V~c~ia Vi~s~s. Nucleic Acids ~sea~ ~: ~35-3~1. 75. ~r, V., F~, O., and Havot N., (1993). ~ S~h~g for ~ ~n~io~ of the No~ p53 ~tvin. ~nds in ~11 Biolo~ 3, 46~9. 76. Rvism~, D., ~d RotOr, V. (1993). ~e Hclix-loop-H~lix containing transcription factor USF b~ds site-speedily to ~ promoter of ~ p53 rumor supp~sor gene. Nucleic Acids Resv~h 21, 345- 350. 77. Almon, E., Goldfi~er, N., ~pon, A., Schw~ D., L~vin~, A., ~d Rotter, V., (1993). Tvsticul~ Tissu~Sp~ific Expression of the p53 Suppressor g~nv. D~wlopment~ Biology 156, 107-116. 79. ~gimov, N., ~uskof, A., Navot, N., RotOr, V., O~n, M., and Aloni, Y., (1993). Wild W~ but not mu~t p53 c~ exp~ss transcription inhibition in vitro by inte~ering with the bind~g of basal tr~s~iption f~tors ~ TATA motif. Oncog,n~ 8, 1183-~193. 80. Schwa~ D., Goldfinger N., and Rotter, V., (1993). ~p~ssion of p53 prote~ in spe~atogenvsis is confined to ~e te~aploid pachytenv p~y sp~atoc~s. On~gene 8, 1487-1494. 81. Rotter, V., Schwa, D., ~mon, E., Golgfinger, E., ~pun, A., M~ho~r A., Donhower~ L.A., ~d A.J., Levine (1993). Micv with ~uc~ lvvels of p53 exhibit the ~ti~l~ ~t cell d¢gvnera~w syndrome. P~. Nat. Acad of Sci. 90, 9075-9079. 82. Aloni-G~s~in, R., ~-B~, I., Alboum I., Goldfingcr N., and Rotter V., (1993) Wi~ Wpe p53 ~ncfions ~ a con~ol p~te~ ~ the diffe~ntiation p~hways of tho B-coil ~vagv. Oncogenv, 8, 3297-3305. 84. Prokocimer, M, ~d Rotter, V. (1994). St~cmre and ~ncfion of p53 in nodal cells ~d ~vir abe~afions ~ c~c,r c~11: pmj~ctlon on ~e hvmaWlogic~ c~ll linvagvs. Blood (~vivw) 84, 2391- 2411. 87. Wolko~cz, R., E~ind, N~., Ronen, D., ~d Rotter, V., (1995). Thv DNA bind~g activiW of wild ty~ p53 is m~ula~d by bloc~ng its v~ous ~tigvnic epitopvs. O~ogene 10, 1167-1174. 88. Eie~nbe~, O. Faber-El~n A., Gottlieb, ~, O~n, M., Rotor, V., ~d Sch~, M., (1995). Divot involvement of p53 in pmgmmm~ ~11 d¢a~ of olig~vnd~cytvs. EMBO J. 14, 1136-11~. 89. ~oni-Gfinst~in R., Schw~ D., ~d Ro~er, V., (1995). A~umulation of Wild ~pe p53 P~tvin, Upon ~I~ti~, ~c~ a ~ Dependent ~muno~obulin Kappa Light Ch~ G~no Exp~mion. E~O J. 14, 1392-1401, 90. Wo~owic~ R., PeI~, A., EI~ H.B., and Rott~, V., (1995). ~ aug~nted DNA bi~g activiw of ~ ~temafiw splic~ C'-~nal p53 is block~ by ~ spliced p53 p~min. P~c. Nat. Acad. of S~nce USA ~, 6~2-6~6. 91. Peller, S, ~, me~c ~ mm~. MoI~ul~ ~inog~sis. in p~ss 92. ~, ~., Go~fmg~ N., ~d ~r, V., spot-l, a p53 ~u~ a ~n 2

Sesrch f~r the normal function(s) of the p53 protei~ ROLE of wild type p53 in cell dlfferentiat±on and • proliferation DMIO0,O00 No overlap tnd~ste tho to~d ~nnu~ funds ~v, dlablo to you tl~s y~r ~or'all r~e~c~ ' p~oj~om under you.r eupervision. ~" Total A~ Annu~ | DM. 180,000 f~)fJ,44c~g

Induction of ~ cells to undergo apoptosis is determined by high levels of wild type p53 protein whereas their differentiation is mediated by lower p53 levels. Dvora Ronen,1 Dov Schwartz,2 Yael Teitz} Naomi Goldfinger2 and Varda Rotter2. 1Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 and 2Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel Running title: p53 -dependent myeloid ceil differentiation and apoptosis Key words: HL-60; apoptosis; differentiation; p53 *Corresponding author:. Varda Rotter Ph. D. Department of Cell Biology Weizmann Institute of Science Rehovot, Israel 76100 TEL: 972-8-343225 FAX: 972-8-344125 E-mail: LCROTTER@~I23¢,_ANN

The observatio~ that wild type p53 may induce ~ to undergo either ap~l~tosis or diffenmtiation raises the question of whether these two events share similar p53-dependent pathways. To evaluate the inte~,latiov~ip bet-,veen these two p53-dependent processes, we have focused our study on the human HL-60, a pro-myeiocytic p53 non-producer cell line in which we have introduced p53 expressior~ and followed induction of apoptosis and differentiation under controlled conditions, p,53 expression was induced in the HL-60 cell line by infection with the recombinant wild typ. e p,53 (p53WT) vaccim'a virus. Viral infection gave rise to cells expressing various levels of wild type p53 protein. High levels of p53 protein induced cells to undergo rapid apoptosis, whereas lower levels of p53 protein induced ceils to undergo cell differentiation at a more moderate rate of kinetics. These results suggest that p53 protein levels may control the determination of whether a given cell should prefer one pathway over the other to exit the cell cycle. Based on these observations, w.e propose that the p53 vaccinia ..virus may be used as a potential vector for cell differential or apoptotic therapy of human primary hematopoetic tumors in vivo. 50544081