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50716971

CTR PROGRF_~S REPORT (for Competiag Renewal Applimtian) 3496 01/01/93 Present Grant number Progress Period from to: Name of Investigator: Subrata Haldar, Ph.D. Title of Original Grant: Molecular and Functional Characterization of bcl-2 Gene Product Abstract of the Specific Aims as stated in the original application: Aim 1- Chaxacterization and purification ofbcl-2 protein expressed in bacculovirus: Aim 1 will focus on the pufifi." ~cation of the bcl-2 protein expressed in bacculovirus to homogeneity. For this purpose FPLC and immunoaffinity techniques will be utilized. We will investigate the biochemical properties with this purified protein. Moreover, the localization of this protein will be ascertained by confocal laser scanning microscopy as well as immunoelectron microscopy. Aim 2 - Studies on the mechanism of inhibition of apoptosis by bcl-2. Aim 2 will be directed towards finding the mechanism by which in bcl-2 inhibits apoptosis in B lymphocytes. For this purpose, coimmunopreeipitation as well as erosslinking studies will be undertaken using antibodies against putative associated proteins and bel-2 molecules. Moreover, immunoaffinity columns prepared with bcl-2 antibody will be used to copurify these molecules from pre B lymphoeytes having bcl-2 in high abundance. List all publications (author, title and journal) resulting from the current CTR award that contain a printed acknowledgment of CTR supporL For mariuscripts, the designation "in press" means you have a preprint: otherwise use "accepted for publication by .... ", "submitted for publication to..." or "in preparation." Do not list publications that do not acknowledge CTR support. See Appendix I 1. Report of Progress (no more than four (41 oaqes, please.) Staple separately. Do not staple this CTR Progress Report to the Application Form. See Appendix II 2. Abstract of CTR Progress Report lone I1| na_qe onlv.} Include your Name~ Grant number and the Title of your projecL Do not st~%ole this Al:~'t~,_ct of CTR. Progress Report to tim Application Form or to th~ Rel~Ort of Program. See Appendix HI 50716972

Ac,~endix I List of publications resulting.from current CTR Haldar, S., Jena, N. and Croceo C.M. (1995) Inactivation of bcl-2 by phosphorylation. Proe. Natl. Aead. Sci. USA 92( 10):4507-4511. Rinaudo, M.S., Su, K., Falk. L.A.. Haldar, S. and Mufson. R.A. (1995) Human Interleukin -3 (IL-3) modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells. Blood (In Press). Gao, X., Knudsen, T.B., Ibrahim, M.M. and Haldar, S. (1995) Bcl-2 relieves deoxyadenylate stress and suppresses apoptosis in Pre-B leukemic cells. Cell Death & Differentiaton 2:69-78. Haldar, S., Jena, N. and Croce, C.M. (1994) Anti-apoptosis potential of bel-2 oncogene by dephosphorylation. Biochemistry & Cell Biology 72:455-462. Baiocchi, R.A., Rose, M.E., Tan, J.C., Chen, C.C., Sullivan, L., Haldar, S., Monne, M., Seiden, M.V., Narula. S.K., S "klar, J., Croce, C.M. and Caliguiri, M.A. (1995) Lymphomagenesis in the SCID - human mouse involves abundent production of human interleukin - 10. Blood 85(4): 1063-1074. o Haldar, S., lena, N., DuBois, G., Reed. J.C., Takayama. S., Fu, S. and Croce, C.M. (1994) Purification and characterization of the bel-2 protein. Arch. Biochem. Biophys. 315:483-488. Haldax, S., ~ena, N., Coss, R.A., Sedar. A.W., Waschsberger, P.R., Beatty, C. and Croce, C.M. (1994) Cellular localization of the bcl-2 protein and response to glucoeortieoid stress. Cell Death & Differentiation 1:109-115. o Carson, W., Haldar, S., Baiocchi, R.A., Croce, C.M. and Caliguiri, M. (1994) The C-kit- ligand suppresses apoptosis of human natural killer cells through up regulation of bcl-2. Proc. Natl. Acad. Sci. U.S.A. 91:7553-7557. 10. Haldar, S., Negrini, M., Monne. M., Sabbioni, S. and Croce, C.M. (1994) Down regulation ofbcl-2 by p53 in breast cancer cells. Cancer Research 54:2095-2097. Negrini, M., Sabbioni, S.. Haldar, S., Possati, L., Costaggnoli, A., Corallini, A.. Barbarti-Brodano, G. and Croce, C.M. (1994) Tumor and growth suppression of breast cancer cells by chromosome 17 associated functions. Cancer Research 54:1818-1824. Abstracts "Loss of anti-apoptosis potential of bcl-2 oncogene by drug-induced phosphorylation - a key for chemoprevention of cancer." Selected as Speaker at Cambridge Healthtech Institute's Second Annual Conference. Commercial prospects t'or Apoptosis Research held at The Cambridge Merriott. Cambridge, Massachusetts on May 1-3, 1995. "Inactivation of bcl-2 by phosphorylation." Abstract accepted for both Oral and Poster Presentation at IBC's International Conference on Apoptosis-Exploiting Cell Death for Therapeutic Apoptosis held at The Royai Sonesta Hotel, Cambridge, Massachusetts on December 5-6, 1994. 50716973

o o Haldar S, Croce CM: And apopmsis potential of bcl-2 oncogcne by depho-~phorlation. Presented at Tenth lmemafional Symposium on Cellular Endocrinology '°Molecular and Cell Biology of Apoptosis in Development, Disease and Cancer" held at The Holiday Inn, Sunplace Resort. Lake Placid. New York. Septembr.r 29-October 2, 1994 Haldar S, Jcna M, Dubois G. Reed JC, Crocc CM: Purificaiton and characterization of the bcl-2 protein. Presented at 85th AACR Conference held in San Francisco, California, April 10-13, 1994 Carson WE, Haldar S, Baiocchi R, Caliguiri MA: Stem factor inhibits apoptosis in CD56 bright NK cells up-regulation of bcl-2. Presented at 85th AACR Conference held in San Francisco, California, April 10-13, 1994 Baiocchi R, Haldar S, C:aliguiri M: Molecular characterization of EBV-induced solid tumor in SCID mice. Presented at 85th AACR Conference held in San Francisco, California, April 10-13, 1994 Haldar S, Negrini M, Monne M, Sabbionni S, Croce CM: Down regulation of bcl-2 by p53 in breast cancer cells. Abswact accepted for presentation at the symposium organized by Bristol-Myers and Fels Institute at Chestnut Hill Campus of Temple University, Philadelphia, Pennsylvania. April. 1994 Haldar S, Beatty C, Henn N, Coss RA, Sedar AW, Wachsberger PR, Croce CM: Cellular localization of the bcl-2 protein. Presented at Cambridge Health Tech. Institute Meeting. Application of Apoptosis Programmed Cell Death , a~uh G~atid Torrey Pines, La Jolla, California Haldar S, Jena N, Coss RA, Sedar AW. Wachsberger PR, Croce CM: Cellular localization of the bcl-2 gene product. Presented at the AACR Special Conference "Cell death in cancer and development" in Cape Cod, Massachusetts, October 17-21,1993. 50716974

.~ppendix ~ CTR PROGRESS REPORT A. Period Covered: January 1, 1993 to present Grant 3496 was last reviewed competitively by CTR Scientific Advisory. committee in September 1992. The project was funded on January 1, 1993. Personnel on the Proiect Subrata Haldar, Ph.D. Janaki Chintapalli, Ph.D. MaW N~u~,en. B.S. Title on Proiect Principal Investigator Postdoctoral Research Fellow Research Technician Service From 01/01t93 06/01/94 10/01/93 Service To Present Present 05131/94 %Effort 50% 100% 100% B. Previous Specific Aims and Progress toward their Achievement The purpose of the research in Grant 3496 was to purify and characterize the bel-2 protein expressed in baculovirus. Studies depicted in Specific Aim 2 were focused to dissect the mechanism of activation of this anti-apoptotic gene. These objectives have been completed [Haldar et al 1994a; 1994b;1994e; 1995a; 1995b1. We also collaborated with other laboratories to study the mechanism of regulation of bcl-2 under the control of eytokines or chemotherapeutics (Carson et al 1994; Rinanudo et al 1995; Gao et al 1995). The most important observation yielded from our research is the discovery of the mechanism of inactivation of bcl-2. These studies could have important clinical application particularly for apoptosis-inducing therapy (Haldar et al 1995a: 1995b). (See Appendix I for detailed list of publications). C, Succint Account of the Published and Unpublished Finding SPECIFIC AIM I: Characteriz~ation and purification of bcl.2 protein expressed in bacculovirus (a) Eleetrot~orated bel-2 0rotein ~rolon~ efll survival The oncogene product, bcl-2, functions as a repressor of programmed cell death, and is a 26 kDa protein with a single predicted transmembrane segment located at the carboxyl terminus. The bcl-2 protein seems to function in different subcellular compartments as evidenced by several biochemical and ultrastructural studies. The present study was performed to purify bcl-2 protein in significant quantities necessary, for structural and functional studies. For this purpose, the bel-2 gene was overexpressed in either baculovirus system or lymphocytes. Initially, attempts were undertaken to purify bcl-2 protein using conventional methods such as ion exchange or gel filtration chromatography. During these purification attempts we determined that bcl-2 protein is highly hydrophobic and prone to aggregation as might be expected for an integral membrane protein. By ion exchange and gel filtration chromatography, this protein could be partially purified. In order to purify bcl-2 to apparent homogeneity, and avoid the aggregation problem, we prepared immunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequence of human bel-2. The antibody was either coupled to CNBy-activated Sepharose 4B or erosslinked into protein A-Sepharose by dimethylpimelimiciate dihydrochloride. Cellular extract equivalent to 108 bcl-2 overexpressing insect cells or lymphocytes was applied to immunoaffinity columns. Approximately, 500 ~tg purified bcl-2 protein could be recovered as estimated by silver staining 50716975

and immunoblotting. Furthermore. purified bcl-2 protein was electroporated into I:h'c-B lymphocytes which do not express this protein in sufficient quantity to delay the on.cot of glucocorticoid induced apoptosis. Following elecu'opomtion of homogeneously pure bcl-2 protein, the cells were found to prolong cell survival in response to glucccorticoids. (For more information please see the enclosed reprint - Haldar et al, Arch. Biochem. Biophys. 315:483-488, 1994). (b) Cellular localization of bcl-2 oro/ein We pertbrmed immuno~lectromicroscopy, immunofluorescence and subcellular fracdonation studies of insect cells (Spodopetra frugipcrda or SFg) infected with recombinant baculovirus containing bcl-2 eDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in prc-B cells carrying a bcl-2 gcno activated by t(14:18) chromosomal translocation. By immunogold electron microscopy, the localization of bcI-2 was found to be at several inrraccllular sites including nuclear membrane, endoplasmic reticulum, mitrochondria and plasma membrane. Immunofluorescencc studies revealed the presence of the bcl-2 product throughout the cytoplasm whereas biochemical fractionation studies indicated similar pattern as observed by electron microscopy. Our investigation clearly indicalcs the localization of the bcl-2 product at several intracellu]ar sites. Moreover, studies worc also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in distribution of bcl-2 protein were observed, interestingly, aggregated patches of gold labeled bcl- 2 particles were found to be much higher under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization neccssaty to prevent apoptosis. (For more information please see the enclosed reprint - Haldar etal, Cell Death & Differentiation I:I09-I 15, 1994). SPECIFIC AIM Ih Studies on the Mechanism of Inhibition of Apoptosis by bci-2 (a) Down-regulation of bcl-2 by p53 Loss of function of human suppressor gone p53 has been implicated in 75% ofall human cancer. The p53 protein resides primarily in the nucleus and binds to specific DNA sequences. Tumor suppression contributed by p53 is thought by at least two mechanisms: arrest of cell poliferation and induction of cell death through apoptosis. The mechanism by which p53 induces apoptosis is still unknown. Interestingly, wildtype p53 induces apoptosis whereas mutant p53 inhibits the process. In order to understand whether any correlation exists between bcl-2 and p53, we analysed several human breast cancer cells for their expression. We found an inverse correlation between the expression of the two proteins. The results sugges~d that mutant p53 could substitute for bcl-2 function in breast cancer cells and that could also down regulate bcl-2 expression. Moreover, we observed that overexpression of mutant p53 (Mut 175) in MCF- 7 cells could induce down-regulation of bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gone does not contain the negative regulatory element responsible for down regulation. If this mechanism will be proved for the wildtype p53 allele, it may disclose a possible mechanism t'or p53 induced apoptosis: down regulation of bcl-2. (For further information please see the enclosed reprints: (Haldar eral, Cancer Res. 54:2095-2097, 1994; Negrini etal. Cancer Res. 54:1818-1824. 1994). (b) The c-kit ligand suppresses apoptosis of human natural killer cells through the up regulation of hcl-2 The bcl-2 protein plays a cemral rol~ in the regulation of programmed cell death in a variety of tissues, and is pivotal to the survival of lymphocytes in vivo. The growth factors responsible for survival of normal lymphocytes ate unknown, but are likely to maintain viability in part through the regulation of bcl-2 expression. A subset of human natural killer (NK) cells (CD3-CD56bright), are unique among lymphocytes in their constitutive expression of c-kit, a Lvrosine kinase cell surface receptor which binds c-kit ligand (KL). Alon~, KJ., does not promote 50716976

proliferation or further differentiation of CD56brig~t NK cells. ~,Vc now report that, in the absence of serum or additional growth factors. KL prevents apt~ptosis of culture CD56bright NK cells as assessed by DNA fragmentation studies, and maintains viability, as measured by biologic responses (i.e. proliferation and cytotoxieity) to the subsequent addition of other eytokines. Furthermore, we demonstrate that KL induces CD56bright NK cells to express the bcl-2 protein. In the presence of anti-c-kit antibody, the tyrosine kinase inhibitor genistein, or bcl-2 antiscnse, the protective effect of KL on the su~,ival of CD56brigt~t NK cells is dramatieaily reduced. These data demonstrate that the binding of KL to its b'rosine kinase receptor results in the upregulation of bcl-2, thereby preventing apoptosis in this subset of normal human lymphocytes. As soluble K.L is plentiful in normal human serum, this survival mechanism may be operative for CD56bright NK cells in vivo (For further information please see the enclosed reprint - Carson et al, Pro<:. Natl. Acad. Sci. USA 91:7553-7557, 1994). c) Inactivation of bci-2 by Phosphorylation Pathway The anti-apoptosis potential of bcl-2 is well established, but the mechanism of bcl-2 action is still poorly understood. Using the phosphatase inhibitor, okadaic acid or the drug taxol, we found that bcl-2 is phosphorylated in lymphoid cells. Phosphoamino acid analysis reveals that bcl-2 is phosphorylated on serine residue{s). Under similar conditions, okadaic acid or taxol treatment leads to the induction of apoptosis in these ceils. Thus it seems that phosphorylation of bcl-2 protein could inhibit its ability to interfere with apoptosis. In addition, phosphorylated bcl- 2 can no longer prevent lipid peroxidation as required to protect ceils from apoptosis. (For more information please see the enclosed reprints - Haldar et al, Biochemistry. & Cell Biology, 72:455- 462, 1994, Halclar et al. Proc. Natl. Acad. Sci. USA 92:4507-4511, 1995). d) Bei-2 relieves deoxyadenylate stress in Pre-B leukemic cells The influence of bcl-2 activity on 2'-deoxyadenosine-induced apoptosis was investigated in human Pre B leukemic cells, stably transfected with bel-2. Children who lack the enzyme adenosine deaminase (ADA) may present ~vith a congenital disorder known as severe combined immunodeficiency disease (SCID). This disease involves the toxic accumulation of adenosine and 2'-deoxyadenosine which are the natural substrates of ADA, and the resulting destruction of immature T and B lymphocytes. The natural toxicity of 2'-deoxyadenosine has been exploited to the benefit of new therapeutic agents for indolent slow growing B-cell lymphoid neoplasms. Our results suggest that one consequence of bcl-2 activity is suppression of 2'-deoxyadcnosine phosphorylation and elevation in the apoptotic target cells. Relief from deoxyadenylate stress imbalances implies a novel upstream site of bcl-2 activity. (For more information please see the enclosed reprint - Gao et al. Cell Death & Differentiation 2:69-78, 1995). e) Co-localization of bcl-2 and R.ras p23 The report on the association of a Ras like GTP binding protein, R-ras p23 with bcl-2 to prevent apoptosis ( Femandez-Sarabia & Bischoff, Nature 366:224, 1993) promoted us to determine its co-localization by using antibodies against bcl-2 and R-ras p23 (a gift of Dr. Lowe, Genentech). Mouse monoclonal antibody against bcl-2 and rabbit polyclonal antibody against R- ras p23 were employed for these studies. Secondary antibodies in the tbrm of FITC anti-rabbit IgG and Rhodamine anti-mouse lgG co-localized bel-2 and R-ras p23 in endoplasmie retieulum and nuclear membrane I Fig. 1 ) 50716977

Moreover we. also dr, te~r.d GTPase activi~ of R-ras p23 in ~r~ immtmoprecipitate of bzl-2 F~gure 1 Co-localization of belo2 ~nd,R-ms p23 by confocal la~er, scanning mi~o~coav 697 cc~s~ B ~s exp~ss~g bel-2~ere subj~ted to du~ ~ofluoms~nee sm~es. Mouse mon~lon~ bel-2 ~fibody (~mb~dge Rese~eh Bighead) ~d rabbit polyelon~ R- r~ p23 amib~y (a Off ~om Dr. ~we, Genenteeh) were used for anfigen-anfib~y r~efion, bel-2 and R- ~ p23 were detee~ by using ~am~e conjugat~ ~fimouse I~ and F~C eonjugamd and rabbit I~ respectively. Photogaphs were t~en using Biorad eonfoe~ mi~oseope at ~e ~stimte fac~. ~ft - ~TC conjugated R-ras p23 ~ght - Rh~amine eonjugated bel-2. Nom the ~C and Rh~amine sign~ ~e deteeted ~ • e same loemion of the cell (presented by ~ows). eoeo Figure 2 Co-immunopreeipitation of GTPase activity. . of R-ms p23 by bel-2 antibody in several cell membranes To measure GTPase activity in the immunopmcipitate of bcl-2 monoclonal antibody, ~.32p GTP labeled membranes isolated from lymphoid cells were immunopreeipitated with specific monoelonal antibody against bcl-2 and protein A - Sepharose beads (Sigma). Immunoprecipitates were boiled for 5 minutes in 1% SDS and 20raM EDTA at 65"C (Trahey and McMoriek, Science 238:542, 1987) and 5~tl of supematant was chromatographed on PEI cellulose in IG-I2P04 pH 3.4. Publications: A total of 10 papers have been published (Appendix I). Among them PNAS paper "Inactivation of bcl-2 by phosphorylation" has been considered as extraordinary quality and has been rated within the top 10% of the papers published in this field (comments of the contributing members of National Aeademy of Science enclosed). Nine abstracts have b~n published till last review (Appendix I). 50716978

A_~_~enclix_ ITI Almptosis is a widespread process invol~'ing chromatin cleavage at 180-200 ba.~ pair intervals, nuclear cond~nsauon, cellular fragmentation and phagocytosis. It may lag activated by a wid~ range of hormones, growth factors and cytokines, and there is growing evidence the efficiency of certain anti-cancer agents is related to the intrinsic propensity of target cells to respond to these compounds by apopmsis. Bcl-2 is a 26 kilodalbon membrane oneoprotein which is unique in it's ability to suppress apoptosis. Overexpression of p26 bel-2 is the primary lesion in many cases of follicular lymphoma and the problem arises when the distal arm or chromosome 18 translocals from a breakpoint site within the bcl-2 locus to the somatic recombination site of the immunoglobulin heavy-chain locus on chromosome 16. The t(14:18) translocation deregulation bel-2 expression by placing it under the control of Igbr gene regulatory clcmems. Numerous studies have examined the consequences of bcl-2 overexpression in stable transfccted cell lines, transgenie mice and antisense deletion, with the common outcome being altered cell survival. The global nature of this response is especially striking as the diversity of apoptotie stimuli which are blocked by bel-2 includes radiation, growth factor withdrawal, glucocylicoids and multiple chemotherapeutic agent. During the tenure of this project, bcl-2 was overexpressed in baculovirus and lymphoeytes and purified to homogeneity by using immunoaffinity chromatography. Eleetxoporated bcl-2 protein in lymphoid cells could protect cells from glucoeortieoid stress. Moreover, using ultrastructural immunofluoresccnce and subcellular fractions approaches, we concluded that bcl is localized at several intracellular sites. This observation is concordant with that found by other investigators. Another report by us suggested that mutant p53 could substitute for bel-2 function in breast cancer cells by down regulating bcl-2 expression. This mechanism was alone proved in the case of wild type p53 allele, reflecting a possible mechanism tbr p53 induced apoptosis: down-regulation of bcl-2. Our recent observation suggest that dephosphorylated bcl-2 may be a molecular determinant of cell survival. Balanced bcl-2 phosphorylation/dephosphorylation may play an important role in the anti-apoptosis potential. Studies with phosphatase inhibitor indicate the bcl-2 protein can be phosphorylated. Phosphorylation pathway leads to inactivation of bel-2 as evidenced by spontaneous apoptosis in bcl-2 overexpressing cells. Several cell lines expressing differential amounts of bcl-2 protein were treated with phosphatase inhibitor or drug taxol. Most of them underwent apoptosis along with phosphorylation of bcl-2, except lymphoblastoid B cells. Strildngly, in these lymphoblastoid B ceils, we could not detect modified bcl-2 protein following Okadaic acid or taxol exposure. More interestingly, pro-B cells isolated from ALL-1 patients exhibited modified forms of bel-2 protein even without treatment of phosphatase inhibitor. Untreated ALL1 ceils demonstrate significant number of apoptotie cells in comparison to alI other cell lines tested. Treatment with phosphatase inhibitor let to severe apoptosis of these cells along with hyperphosphory, iation of bcl-2. We co-immunoprecipitated another 35KDa protein with bel-2 in phosphatase inhibitor treated cells. Phosphoamino acid analysis suggest that both bel-2 and 35KDa proteins are phosphorylated on serine residues, bcl-2 is known to block lipid peroxidation to prevent apoptosis. Our experiments demonstrate that phosphorylated bcl-2 can no longer prevent lipid preoxidation associated with cell death. In summary., our studies establish a strong correlation between bcl-2 phosphorylation and apoptosis after treatment with okadaie acid and taxol. Our observations could have important clinical implications particularly for apoptosis inducing therapy. 50716979