This major international apoptosis conference in Asia intends to bring scientists from different parts of world to present and discuss most current advances in apoptosis research. The topics to be covered include: lymphocyte cell death, apoptosis mechanisms, developmental/physiological cell death, extracellular matrix and cell death, cell death and cancer, biochemistry and molecular biology of cell death genes and anti-death genes.

National Institutes of Child Health and Development, National Institutes of Health
National Institutes of Environmental Health Sciences, National Institutes of Health

American Red Cross
University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
Shanghai Research Center of Life Sciences, Chinese Academy of Sciences
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

Session I
Session II
Session III
Session IV
Session V
Session VI Apoptosis and Tumorigenesis
Apoptosis Signaling
Apoptosis and Immune Systems
Apoptosis in Development and Disease
Apoptosis Mechanisms
Apoptosis and Physiology

Pro-apoptotic BAX and BAK mediate p53-independent suppression of tumorigenesis
Eileen WHITE (267)
Mechanism of Smac in regulating cell apoptosis and its implication in cancer chemotherapy
Jian ZHAO, Jun JIN, Meng Chao WU, Ya Jun GUO (268)
Role of genetic abnormalities of PTEN and the phosphatidylinositol 3 kinase pathway in breast and
ovarian cancer tumorigenesis, prognosis and therapy
Gordon B MILLS, Yi Ling LU, Xian Jun FANG, Hong Wei WANG, Astrid EDER, Mu Ling MAO, Ramona SWABY, Kwai Wa CHENG, Kathy SIMINOVITCH, Elise KOHN, David STOKOE, Wen Lin KUO, Robert JAFFE, Joe GRAY (269)
Tumor suppressor gene PTEN affects anoikis via both PKB and FAK in human lung carcinoma cell Lines
Qun Ying LEI, Li Ying WANG, Zhen Yu DAI, Xi Liang ZHA (270)
Proteomic analysis of apoptosis triggered by inhibition of ubiquitin-proteasome pathway in Mo7e leukaemic cells
BF JING, HX WANG, J WANG, K HE, M YU, MR HU, KH WEI, SC YANG, BF SHEN, XM ZHANG (271)
Surprising functions of IAPs
Jonathan D ASHWELL (272)
Permanent liver allograft survival induced by transfusion of apoptotic donor splenocyte without immunosuppression
Yi GAO, Shuang WANG,Yufang SHI, Er Wei SUN (272)
Induction of apoptosis in human T leukemic cell line CEM-6T by tripterine: comparison with arsenic oxide and hydroxyl camptothecin
Deng Hai ZHANG, Xue Qiang HUANG, Chun Xin YANG, Wan Zhang QIN, Xian Tao KONG, Yi Xiao BAO, Ting Xuan WEI, Min Shu XU, Qin Heng HUANG (273)
The in vitro antiapoptotic effect of lithium chloride in mouse thymocytes
Yan Mei TIAN, Xiao Fang JIANG, Yao Ren DAI (274)
Traditional Chinese herbal formulate Jin Gui Shen Qi Wan affects the protein levels of Bcl-2 family members and suppresses apoptosis in human lymphocytes
Deng Hai ZHANG, Chanquan LIN, Xue Qiang HUANG, Yi Xiao BAO, Ting Xuan WEI, Qin Heng HUANG (274)
Apoptosis of breast cancer cell MCF-7 induced by norcantharidin (NCTD)
Jian Li SANG, Xiao Zhu LI, Hai Jiang ZHANG (275)

Cytotoxic T cell-induced apoptosis
Chris BLEACKLEY (276)
Akt and Bcl-x_L are independent regulators of the mitochondrial cell death pathways
David R PLAS, Jeffrey C RATHMELL, Craig B THOMPSON (276)
Mechanisms of caspase activation and inhibition during apoptosis
Yi Gong SHI (277)
Transcriptional regulation of IL-3-Dependent survival responses
Jeffrey JY YEN, Yung Luen YU, Yun Jung CHIANG, Wannhsin CHEN (278)
Selection and study on minoligand of Acanthamoeba which could induce tumor cells to apoptosis by phage display technique
Xue Liang GAO, Min QIAN, Zhong Liang ZHOU, Ping ZHANG, Meng YU (279)
Integration of TNF-a signaling: Crosstalk between caspases, IKK and JNK
Anning LIN (280)

Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL-null mice
S-E LAMHAMEDI-CHERRADI, Shi-Jun ZHENG, Kimberly A MAGUSCHAK, Jacques PESCHON, Youhai CHEN (281)
Required components of commitment to apoptosis in thymocytes
Noriko TONOMURA, Barbara A OSBORNE (281)
Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: Role of apoptosis in T helper subset differentiation
Xiao Ren ZHANG, Satish DEVADAS, Li Ying ZHANG, Li LI, Achsah D KEEGAN, Yu Fang SHI (282)
Signaling for inducible Fas-resistance in primary B lymphocytes
Thomas L ROTHSTEIN (282)
Failure to disengage PI-3K pathway signaling confers anti-IgM resistance to growth arrest and apoptosis in the CH12 B-Cell Lymphoma
Gregory B CAREY, Laura TONNETTI, David W SCOTT (283)
Transfusion of donor apoptotic splenocytes prolongs cardiac allograft survival in absence of Immunosuppression
Er Wei SUN, Yi GAO, Jian Feng CHEN,Yu Fang SHI (284)
Peripheral clonal deletion of MBP-specific T cells by intravenous autoantigen: rapid reversal of ongoing, progressive experimental autoimmune encephalomyelitis
Guang Xian ZHANG, Divina CALIDA, Thomas T LIU, Elvia S VENTURA, Youhai CHEN, AM ROSTAMI (284)

The Role of Bax and Bak in cell death in the nervous system
Tullia LINDSTEN, Wei Xing ZONG, Jeffrey GOLDEN, Alan V WHITMORE, Marian HARRIS, Martin C RAFF, Craig B THOMPSON (285)
Thyroid hormone induced apoptosis during amphibian metamorphosis
Yun-Bo SHI (286)
Mitochondrial mechanism of cardiomyocyte apoptosis induced by stress
Ling Jia QIAN, Hui Rong REN, Wan Ying WANG, Jing Bo GONG, Xue Li SONG (286)
Breakdown of chromosomal DNA during apoptosis and phagocytosis
Shigekazu NAGATA (287)
Virus infections and apoptosis: Lessons from HIV
Xiao Ning XU (287)
A novel mediator of the amyloid-b neurotoxicity in Alzheimer's disease: Ubiquitin conjugating
enzyme E2-25K/Hip-2 as upstream of caspase-12
Yu Hyun SONG, So Young KIM, Yeon Mi HONG Joo Yong LEE, Yung Joon Yoo, Yong Keun JUNG (288)
PML gene, arsenic and apoptosis of leukemic cells
Guo Qiang CHEN (288)
Activation of p53 in gastric cancer cells by tripchlorolide specifically induces apoptosis
Jia Rui WU (289)

DNA fragmentation in apoptosis and homeostasis
Ming XU, Jian Hua ZHANG (290)
Cloning and identifying of a novel protein binding with death domain of the death receptor 4
Xiao Ling LI, Yan Xin LIU, Shi Lian LIU, De Xian ZHENG (291)
Regulation of the subcellular localization of TRAF2 by TRAF1 reveals mechanisms of TRAF2 signaling
Yongwon CHOI (291)
Characterization of a novel proapoptotic protein, map-1, that associates with bax through its multiple bcl-2 homology domains
Kuan Onn TAN, Shing Leng CHAN, Nai Yang FU, Karen MEI Ling TAN, Karen SY YEE, Victor C YU (292)
Fas ligand: Can it ever be tamed?
RC DUKE, K HANCE, J SUN, D CHAN, SJ MEECH, B PIETRA, D NELSON, D BELLGRAU (292)
Cloning and identification of apoptosis specific DNase inhibitor IXAD in Xenopus egg extract
Zhi Gang LU, Chuan Mao ZHANG, Zhong He ZHAI (293)
N-Myc downstream-regulated gene 2 (NDRG2) is a negative regulator of cell growth and its expression is decreased in cancers
Li Bo YAO, Yan Chun DENG, Shu Jun LI, Ling CHAU, Wo-Shing AU, Xin Ping LIU, Ji WANG, Jian LI, Fu Yang LI, Shao Ping J, Hsiang Fu KUNG, Suet Yi LEUNG, Marie CM LIN (293)
Selective induction of apoptosis in K562 cells by a BCR-ABL tyrosine kinase inhibitor, STI571
Xue Mei SUN (294)
The protective role of heat shock protein against cardiomyocyte apoptosis by mitochondrial pathway
Wei Min XIAO, Bi Mei JIANG, Yong Zhong SHI, Mei Dong LIU, Dao Lin TANG, Xian Zhong XIAO (294)
AMID, an AIF homologous protein, induces caspases-independent apoptosis
Min WU, Zhong He ZHAI, Hong Bin SHU (295)
15-deoxy-¦¤12,14-prostaglandin-J2 induces apoptosis in ECV304 endothelial cells by inhibiting NF-kB and AP-1 activation pathways
Dan Dan CHEN, Yu Gang DONG (295)
How Bax sequesters its hydrophobic membrane anchor to regulate both subcellular localizaton and dimer formation
Motoshi SUZUKI, Seon Yong JEONG, Richard J YOULE (296)

Phosphoinositides as ligands of PHD-fingers: implications for chromatin remodeling
Or Gozani, Philip Karuman, Seth Field, Hong ZHU, James CHA, Jennifer VILLASENOR, Vikram R RAO, Joan S BRUGGE, Lewis C CANTLEY, Jun Ying YUAN, Cheryl L BAIRD, Bharat MEHROTRA, Jian CHEN, David G MYSZKA, Colin G FERGUSON, Glenn D PRESTWICH (297)
Transforming growth factor-b1 suppresses serum deprivation-induced apoptosis via activation of ERK1/ERK2 and the inhibition of p38 activity and ceramide production
He Hua CHEN, Sheng ZHAO, Jian Guo SONG (298)
Activation/Inactivation of ion channels and pumps during apoptosis: a necessary role in the apoptotic volume decrease
John A CIDLOWSKI, Carl D BORTNER, Maria Gomez ANGELATS, Alyson SCOLTOCK, Nina Storey, David ARMSTRONG, Mona RAZIK (299)
Human Lung Fibroblast Express Acetylcholinesterase During Apoptosis in vitro
Xue Jun ZHANG, Qi Huang JIN, HY HE, H JIANG, Lei YANG, ZQ JIANG, YF CHAI, Yu Fang SHI (299)
Neuroprotection against apoptosis: what has it got to do with lithium, a mood stabilizing drug
De Maw CHUANG (300)
RNA splicing and SR protein phosphorylation under stress in acute myeloid leukemia (AML)
Tien Sheng HUANG, Karin FLADMARK, Stein Ove DOSKELAND, Johan R LILLEHAUG (301)
Thx cells: a new subset in T helper cells, are they born to die
Satish DEVADAS, Yufang SHI (301)
Integrating immunome, tissue microarray and human-human hybridoma technology to develop natural human apoptosis-related antibodies
Jian Ni, Lei LIU (302)
The role of prostaglandins in regulating vascular smooth muscle cell survival
Rung Chi LI, Tereza CINDROVA, Tai Ping FAN, Jeremy SKEPPER, Lynda SELLERS (303)
Essential role of voltage-dependent anion channel (VDAC) in mitochondrial permeability transition and cytochrome C release induced by arsenic trioxide
Yan Hua ZHENG, Rong SHI, Chu Sun JIANG, Hai Jing JIN, Jian Jun CHEN, Hong TANG, Quan CHEN (304)

Pro-apoptotic BAX and BAK mediate p53-independent suppression of tumorigenesis

Howard Hughes Medical Institute, Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Cancer Institute of New Jersey, 679 Hoes Lane, Room 140, Piscataway, NJ 08854, USA
E-mail: ewhite@cabm.rutgers.edu

BAX and BAK are essential regulators of apoptotic signaling through mitochondria in mammalian development and in response to cytotoxic stimuli. To investigate the role of BAX and BAK in transformation and tumorigenesis, primary baby mouse kidney epithelial cells (BMKs) from wild-type, BAX, BAK and BAK and BAK deficient mice were transformed by adenovirus E1A and dominant-negative p53 (p53DD). While E1A alone transforms p53 deficient BMKs, transformation of BAX and/or BAK deficient BMKs still required inactivation of p53. Since BAX and BAK are dispensable for p53 to suppress transformation, but their deficiency causes a profound defect in apoptosis, we evaluated their contribution tumorigenesis. E1A plus p53DD transformed wild-type, BAX, BAK, or BAX and BAK deficient BRK cell lines were injected subcutaneously into nude mice and evaluated for tumor xenograft formation. None of the wild-type BMK cell lines formed tumors, nor did E1A alone transformed, p53 deficient BMK cell lines. Thus expression of E1A and inactivation of p53 was sufficient for transformation but not tumorigenesis. In contrast, all BAX and BAK deficient BMK cell lines formed highly invasive carcinomas over a wide range of injected cell numbers. BMK cell lines deficient for either BAX or BAK were also tumorigenic but only when heterozygous for the remaining BAX or BAK allele, the expression of which was lost in the resulting tumor. Thus, BAX and BAK function to suppress tumorigenesis, and their loss can be selected for in vivo. Furthermore, suppression of tumorigenesis by BAX and BAK is independent of the functional status of p53, suggesting that restoration of apoptosis to tumor cells that have inactivated mitochondrial death signaling may provide an advantage in anti-cancer treatments.

Mechanism of Smac in regulating cell apoptosis and its implication in cancer chemotherapy

International Joint Cancer Institute and Eastern Hospital of Hepatobiliary Surgery, The Second Military Medical University, Shanghai 200433, China
^*E-mail: < yguo@unmc.edu > < ewhite@cabm.rutgers.edu >

The Second mitochondrial-derived activator of caspase (Smac), a mitochondrial protein, plays an important role in inducing cell apoptosis by activating caspases. After receiving apoptotic signals, mitochondria releases cytochrome c to induce the formation of caspase-9 activating apoptosome and releases Smac to counteract the inhibitory activity of IAPs. Mature Smac binds to human IAP family members and relieves their inhibition of caspases. In this study, the human osteosarcoma cell line Saos-2 was used to establish the apoptosis model. Smac expression during cell growth and its biological effects in inducing cell apoptosis was explored. In addition, the interactions between Smac and other apoptotic regulatory proteins such as P53, Bcl-2 and Bax was further investigated. Anti-tumor effect of Smac both in vitro and in vivo was tested in this tumor model. To investigate the biological functions of Smac on Saos-2 cell proliferation, vectors encoding smac and anti-sense smac were transfected into the Saos-2 cells. The change of cell cycle, cell growth curve and the sensitivity to etoposide were observed. The results demonstrated that Over-expressed Smac could promote the sensitivity of the cells to chemotherapeutic drugs and there were no significant effects on cell cycle or cell growth. Inhibition of smac expression with anti-sense smac vector could facilitate the cell growth with an increasing cell population in G1/G0 phase, which might contribute to a low sensitivity of the cells to chemotherapy drugs. To investigate the role of Smac in apoptosis, etoposide and TRAIL induced apoptosis models were used. The Smac distribution during apoptosis was detected by western blot. The results showed that Smac is released to the cytosol from mitochondrial during apoptosis. Smac can increases the cell sensitivity to apoptotic signals in both mitochondrial and the TNF apoptotic pathways.

In order to study the relationship between Smac and other apoptosis-regulating proteins such as P53, Bcl-2 and Bax, we investigated the distribution of the cytosol Smac and the apoptotic development of the various transfected Saos-2 cells induced by different apoptotic signals. The results demonstrated that Bcl-2 could block the Smac release to the cytosol in apoptosis process induced by TRAIL and etoposide. However, Bcl-2 had no effect on Ad.smac- induced apoptosis. This clearly indicated that Smac had a function in the down-stream of bcl-2 in apoptotic pathway. Smac was also required in P53-induce apoptosis, which could be inhibited either by over-expression of Bcl-2 to reduce Smac releasing into cytosol or by transfection with anti-sense smac to inhibit the Smac expression. Bax can also enhance the cell apoptosis by regulating Smac release. The data further illustrate the role of Smac in apoptosis.

Because Smac was involved in several steps of apoptotic pathways and was able to turn the apoptotic-resisting cells into apoptotic-sensitive ones, it may have potential application in cancer chemotherapy. To explore the anti-tumor effect of Ad.smac in vitro, replication deficient adenovirus Ad.smac was successfully developed. The anti-tumor effects of Ad.smac on various tumor cells were observed. It has been clearly shown that Ad.smac can effectively induce the death of Saos-2, HepG2, PLC/PRF/5, ACC-2 cell lines in vitro. Ad.smac treatment alone can inhibit the HepG2 growth in nude mice and when combined with 5-FU, a complete tumor regression were obtained. The results suggest that this new strategy may be useful for the treatment of cancer.

Role of genetic abnormalities of PTEN and the phosphatidylinositol 3 kinase pathway in breast and ovarian cancer tumorigenesis, prognosis and therapy

Gordon B MILLS, Yi Ling LU, Xian Jun FANG, Hong Wei WANG, Astrid EDER, Mu Ling MAO, Ramona SWABY, Kwai Wa CHENG, Kathy SIMINPVITCH, Elise KOHN, David STOKOE, Wen Lin KUO, Robert JAFFE, Joe GRAY

Department of Molecular Therapeutics, University of Texas M.D. Anderson Cancer Center, Box 317, 1515 Holcombe Blvd., 77030, Houston, TX, USA
E-mail: gmills@mail.mdanderson.org

Breast and ovarian cancers exhibit several similar epidemiologic, genotypic and phenotypic characteristics suggesting that similar underlying genetic defects may contribute to the development of both tumor types. Phosphatidylinositol 3 kinase (PI3K) and the PTEN tumor suppressor gene product phosphorylate and dephosphorylate the same 3' site in the inositol ring of membrane phosphatidylinositols. Germline mutations in the PTEN tumor suppressor gene are causative of the Cowden's breast cancer predisposition syndrome and PTEN is frequently mutated or expressed at decreased levels in sporadic breast cancers. PTEN is also frequently mutated in gliomas, prostate cancer, endometrioid ovarian cancer and endometrial cancers and present at decreased levels in multiple other cancers. In contrast, genomic, RNA and protein amplification of multiple components of the PI3K pathway, in the absence of mutations in PTEN, is a hallmark of serous epithelial ovarian cancer. Amplification of components of the PI3K is also frequently observed in cervix, head and neck, lung and pancreatic cancers. Further, a mutant transforming PI3K regulatory subunit has been found in lymphomas and the downstream target, AKT, has been incorporated into a transforming retrovirus. Indeed, an ability to link to PI3K is required for transformation by multiple oncogenes including polyoma middle T, RAS, and members of the HER2 family of cell surface receptors. Mutation of PTEN, expression of PTEN, and amplification of genomic sites containing components of the PI3K pathway have been demonstrated to confer a prognostic load indicative of an important role in patient outcome either through altering the pathophysiology of the disease or by altering response to therapy. The PI3K pathway is a major target of the HER2 family of transmembrane receptors, which contribute to breast and ovarian tumorigenesis. Mutation of PTEN and amplification of PI3K signaling have the potential to alter the function of steroid hormone receptors, BRCA1, p21, p27 and p53, all of which can contribute to the development of breast and ovarian cancers. Thus the PI3K pathway has the potential to contribute to oncogenesis and potentially, selectively to the initiation or progression of breast and ovarian cancer. Abnormalities in PTEN and PI3K in breast and ovarian cancer cells, respectively, suggest that different genetic events may contribute to a similar phenotype. However, amplification of components of the PI3K pathway or mutation in PTEN, while both contributing to dysregulation of the PI3K pathway, has subtly different biochemical consequences. Mutation of PTEN increases baseline signaling and the duration of agonist induced signaling through the PI3K pathway rendering cells autonomous from their environment. In contrast, amplification of components of the PI3K pathway increases the sensitivity of cells to exogenous ligands, resulting in a phenotype that is not cell autonomous but rather dependent on interactions with the cell environment. The resultant activation of the PI3K pathway in both breast and ovarian cancers contributes to cell cycle progression, decreased apoptosis, increased invasiveness, increased production of neovascularization factors and increased metastatic capabilities. Strikingly, both ovarian and breast cancer cells with abnormalities in the PI3K pathway are selectively sensitive to pharmacological and genetic manipulation of the PI3K pathway at multiple levels, making molecular therapeutics targeting the PI3K pathway particularly attractive approaches in these diseases. We have explored the effects of small molecules targeting PI3K and FRAP/TOR, as well as gene therapy approaches targeting PI3K, PDK1, and PTEN on the in vitro and in vivo growth of tumor cells. Dependent on the environmental context, these approaches can induce a cell cycle arrest at G1 or G2/M followed by apoptosis. Inhibition of the PI3K pathway renders cells selectively sensitive to anoikis, apoptosis that occurs following disruption of epithelial stromal interactions, suggesting that inhibition of the PI3K pathway may be particularly effective in preventing metastases. Strikingly, cells with a competent PI3K pathway are generally resistant to the effects of inhibitors of the PI3K pathway. Compatible with this model, enforced amplification of signaling through the pathway renders cells sensitive to small molecules targeting downstream events. While abnormalities in PI3K or PTEN are strong predictors of responses to molecular therapeutics targeting the pathway, they are neither sufficient nor required for sensitivity. Inhibition of the PI3K pathway in vivo decreases tumorigenesis, proliferative rates, and alters vascular permeability while increasing apoptosis and sensitivity to chemotherapeutics. Several inhibitors of the PI3K pathway have limited toxicity in animal models suggesting that an adequate therapeutic index may be present to allow use in human cancer patients. Most of the drugs and gene therapy approaches targeting the PI3K pathway are at the proof of concept or animal model stage. However, the rapamycin analog, CCI 779, which targets the PI3K pathway at the level of TOR or FRAP, is currently in phase I and phase II trials. Only a portion of tumors from patients with any specific tumor type, including breast and ovarian cancer, exhibit abnormalities in the PI3K pathway. Further, abnormalities in the PI3K pathway can occur at multiple levels. The success of molecular therapeutics targeting the PI3K pathway will likely be dependent on an ability to identify patients with abnormalities in the pathway. As abnormalities can occur at multiple levels, it will be necessary to develop global methods to assess functionality of the complete PI3K pathway in patient tumors to determine which patient may respond to drugs targeting the PI3K pathway.

Tumor suppressor gene PTEN affects anoikis via both PKB and FAK in human lung carcinoma cell lines

Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Boichemistr and Molecular Biology, ShanghaiMedical College, Fudan University, Shanghai 200032, China

PTEN was identified in 1997 as a new tumor suppressor gene on human chromosome 10q23, deletions and mutations of this gene were associated with a variety of human cancers such as glioblastoma and prostate cancer. In this study Northern blot analysis was carried out and one major band at 2 kb region was observed in all 8 HLCC samples, consistent with previous reports. The result showed that the PTEN gene were expressed and its mRNA level similar in all cell lines tested. To determine whether the PTEN mRNA level reflects the parallel level of protein, the level of PTEN protein was examined by Western blot.PTEN protein level was high in H₄₆₀ and detectable in A₅₄₉, A₄, A₇, L₁ cells, not detectable in 95C, 95D, A₁ cells. Interestingly, PTEN mRNA expression was close each other in 8 HLCC lines, but protein levels were quiet different. The mRNA stability was determined in H₄₆₀ and 95D cell lines. The PTEN mRNA was much stable in H₄₆₀ than in 95D. This result indicated that PTEN expression might be regulated post-transcriptionally or translationally. Anoikis can be induced in H₄₆₀ cells with SFM and loss of attachment. DNA ladder can be observed in H₄₆₀, while 10% FBS can prevent it from conducting anoikis; no DNA ladder can be detected in the other HLCC lines without or with serum medium. Protein kinase B (PKB) and focal adhesion kinase (FAK) are well-known anti-apoptotic factors. We observed that PTEN protein level correlated oppositely with the ratio of PKB-phospho/PKB in 8 HLCC lines and associated with a reduction of FAK in H₄₆₀. These results suggested that PTEN involved in the process of anoikis and its gene expression was regulated post-transcriptionally or translationally in some HLCC lines.

We thank Dr. Qiang Yu from Bosten University for kindly providing the PTEN plasmid and Dr. Jiu-xian Feng and Hui-Fang Sha from the Chest hospital (Shanghai) for kindly supporting in lung carcinoma cells culture. This work is supported by grants from National Nature Science Foundation of China (No. 39970388) and Shanghai Municipal Government Science and Technology Committee (No. 00JC14042).

Proteomic analysis of apoptosis triggered by inhibition of ubiquitin-proteasome pathway in Mo7e leukaemic cells

Institute of Basic Medical Sciences, National Center of Biomedical Analysis, 27 Tai-Ping Road, Beijing 100850, China
E-mail: xmzhang@nic.bmi.ac.cn.

The ubiquitin-proteasome pathway is critical for the degradation of short-lived proteins in eukaryotic cells, and inhibition of this pathway could induce apoptosis in human leukaemic Mo7e cells. We analyzed the proteomic response of Mo7e cells by inhibiting the ubiquitin-proteasome pathway with . Among 72 protein spots showing significant changes in expression on 2-D protein gels, 4 of them were strongly increased. They are identified as Rho GDP dissociation inhibitor (GDI) b protein, profilin 1, adenylate kinase isoenzyme 2, and eIF-5A as determined by peptide mass fingerprinting(PMF) using MALDI-TOF-MS and peptide sequence tag(PST) using nano-ESI-MS/MS(Q-TOF2, Micromass). Additional two spots were also identified as Rho GDI, which we believe to be due to unknown modifications of this protein. Using a caspase-3-specific inhibitor DEVD-CHO, we showed that up-regulation of profilin 1 and Rho GDI proteins is associated with the activation of caspase-3, which was activated by Z-LLL-CHO. The strong induction of eIF-5A by inhibition of the ubiquitin-proteasome pathway seemes to be inconsistent with the result obtained with apoptotic cells induced by other methods, in which the level of eIF-5A has always been shown to be suppressed. The plausible explanation for this observation is that eIF-5A protein accumulation induced by inhibition of the ubiquitin-proteasome pathway was not processed through the post-translational modification by spermidine and subsequent hydroxylation to form hypusine in mature eIF-5A. which has been shown to be necessary for growth and viability in yeast. Our data seems to be consistent with the hypothesis that inhibition of the ubiquitin-proteasome pathway suppressed the post-modification of eIF-5A and caused the accumulation of the unmodified eIF-5A. Therefore, our proteomic analysis has revealed that AK2, rather than other isozymes of AK (AK1, AK3), was over expressed in apoptosis, a conclusion would have been difficult to reach by other approaches.

This work was supported by grants from National Natural Science foundation of China (No. 30070377), and National High Technology Research and Development Program of China (No. 2001AA233041).

Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
E-mail: jda@pop.nci.nih.gov

The destruction of proteins in proteasomes is regulated at various levels. One of the early rate-limiting steps is ``tagging" of proteins with ubiquitin, an 8 kD molecule that, when present in chains of four residues or more, serves as a signal recognized by proteasomal subunits. Ubiquitin is attached to target proteins by a series of enzymatic steps involving an E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin protein ligase). It is the last enzyme that confers target protein specificity. We have found that members of a family of RING domain containing proteins with anti- apoptotic activity, IAPs, have intrinsic E3 activity toward themselves and other substrates. This activity is induced in thymocytes stimulated to undergo apoptosis, resulting decreased expression of these anti-apoptotic molecules. Two of the IAPs (c- IAP1 and c-IAP2) are part of the TNF receptor (TNF-R) signaling complex. TNF-alpha is a proinflammatory mediator that exerts its biological functions by binding TNF-R-I and -II, which initiate biologic responses by interacting with adaptor and signaling proteins. Among the signaling components that associate with TNF-Rs are members of the Tumor Necrosis Factor Receptor-Associated Factor (TRAF) family. TRAF2 is required for TNF-a-mediated activation of JNK, contributes to activation of NF-kB, and mediates anti-apoptotic signals. The function of IAPs in TNF-R signaling is unknown. We have found that binding of TNF-a to TNF-RII induces ubiquitination and proteasomal degradation of TRAF2. Moreover, c-IAP1 was able to ubiquitinate TRAF2 but not TRAF1 in vitro, and overexpression of wild type c-IAP1, but not an E3-defective mutant, resulted in TRAF2 ubiquitination and degradation in cells. These findings identify for the first time a physiologic role for c-IAP1 and define a echanism by which TNF-RII-regulated ubiquitin protein ligase activity can potentiate TNF-induced apoptosis.

Permanent liver allograft survival Induced by transfusion of apoptotic donor splenocyte without Immunosuppression

¹Organ Transplantation Department, Zhujiang Hospital, First Military Medical University, Guangzhou, China
²Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA
E-mail: ewsun@263.net

Recent progress in apoptosis study has revealed that it is not only a process to quietly eliminate excessive cells, but also plays important roles in immune regulation. In the present study, we investigated whether donor apoptotic cell can be used to induce transplant immune tolerance. Dexamethasone (Dex, 3mg/kg/day for 3 days) was injected intraperitoneally into adult Sprawn-Dowley (SD) rats. Splenocytes from those dex-treated rats were isolated and transfused into Wistar adult rats. One week later, Sprawn-Dowley liver were transplanted into the Wistar rats received dex-treated splenocytes. Dex treatment often induces apoptosis in 34.9% splenocytes. Transfusion of 5¡Á10⁷ apoptotic donor splenocytes is sufficient to induce a permanent liver graft survival (median graft survival, MST > 350 days) in adult recipient rats without administration of any immunosuppressants. Liver function test and histological analysis further confirmed the lack of rejection. These results demonstrated a novel protocol for inducing transplantation tolerance in adult recipients in absence of the toxic and side effects associated with immunosuppressants. More importantly, the results strongly argue that the status of the cells with which an antigen associate plays a pivotal role in immune responses. Thus, self and nonself immune recognition is not solely determined by the nature or the delivery pathway of an antigen.

Induction of apoptosis in human T leukemic cell line CEM-6T by tripterine: comparison with arsenic oxide and hydroxyl camptothecin

Deng Hai ZHANG^1,2, Xue Qiang HUANG², Chun Xin YANG³, Wan Zhang QIN³, Xian Tao KONG⁴, Yi Xiao BAO⁴, Ting Xuan WEI¹, Min Shu XU¹, Qin Heng HUANG¹

¹ Clinical Research Center, Shanghai East Hospital, Shanghai Tongji University, China
² Department of Traditional Chinese Medicine, Changhai Hospital, Shanghai Second Military Medical University, China
³ Department of Dermatology, Zhongshan Hospital, Fudan University, China ⁴ Clinical Diagnostic Center, Changzhen Hospital, Shanghai Second Military Medical University, Shanghai 200433, China
E-mail: shanghai_-zhang@hotmail.com

In China, the herbal plant Lei gong teng (tryipterygium wolfordii Hook F) is widely used in rheumatic diseases, indicating that some compounds in this plant might have effects on immune cells. We have reported that one of the compound from this plant, tripterine could induce apoptosis in the human mast leukemic cell line HMC-1 ( Chin J of Hematol 20:146,1999). To further explore the potential value of triperterine in the treatment of leukemia, we cultured human T leukemia cell line CEM-6T in vitro with tripterine, or arsenic oxide, or hydroxyl camptothecin at various concentration ranging from 0.0625 mM to 128 mM DNA electrophoresis and annexin V labeling were used to detect apoptosis. The protein levels of Bcl-2, Bax, Bcl-xL, Bcl-xS, Fas, and FasL were also determined in tripterine-treated cells. DNA ladder was revealed in samples treated with any of the three investigated drugs, despite the difference in their effective concentrations. Quantitative analysis showed that the apoptotic rates increased with the dose at 0.125-1 mM in tripterine treated samples, while the necrotic rates became prominent and increased with higher dose. In the samples treated with arsenic oxide, the apoptotic rate increase at 0.5-8.0 mM, exceeding which apoptotic and necrotic rates turned to decreased_. Hydroxyl camptothecin induce increased apoptosis at 0.125-1 mM, exceeding which the apoptotic as well as the necrotic rates unchanged. Bax were increased while Bcl-2 remained unchanged in the samples treated with tripterine at dosage that trigger apoptosis. Bcl-xL decreased while Bcl-Xs increased at first and then decreased. CEM-6T cells expressed Fas but not FasL, and such expressing patterns were not changed by tripterine. Therefore, tripterine, like arsenic oxide and hydroxyl camptothecin, could induce T leukemic cells into apoptosis, though with different dose-dependent effects. Effects of tripterine in inducing apoptosis might be related to readjust the equivalences between the anti- and pro-apoptotic members in bcl-2 family, independent of Fas and FasL. Finally, our results support the potential of using tripterine of its related compounds in the treatment leukemia.

Youth Talent Grant in Science of Shanghai Technology and Science Committee (00QB14007).

School of Life Science and Engineering, Tsinghua University, Beijing 100084, China
E-mail: zhaizhh@mail.tsinghua.edu.cn

The effects of lithium chloride (LiCl) on thymocyte apoptosis induced by dexamethasone (DEX) were investigated. When primary culture of thymocytes was preincubated with 100 mM LiCl before their exposure to DEX, apoptotic cell death induced by DEX was almost completely prevented as determined by both flow cytometric analysis and the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. DNA laddering assay also documented the inhibition of thymocyte apoptosis by LiCl. Furthermore, we found that the DEX-induced increment of caspase-3 activity in thymocytes was completely abolished by LiCl preincubation. Our results showed that lithium chloride may rescue Balb/c mouse thymocytes from apoptosis induced by dexamethasone in vitro and the spontaneous apoptosis in a dose-dependent matter.

Traditional Chinese herbal formulate Jin Gui Shen Qi Wan affects the protein levels of Bcl-2 family members and suppresses apoptosis in human lymphocytes

Deng Hai ZHANG^1,2, Chan Quan LIN², Xue Qiang HUANG², Yi Xiao BAO³, Ting Xuan WEI¹, Qin Heng HUANG¹

¹Clinical Research Center, Shanghai East Hospital, Shanghai Tongji University
²Department of Traditional Chinese Medicine, Changhai Hospital, Shanghai Second Military Medical University
³Department of Paediatrics, Xinhua Hospital, Shanghai Second Medical University, Shanghai 200025, China
E-mail: shanghai_-zhang@hotmail.com

The Bcl-2 knockout mice demonstrated fulminant lymphoid apoptosis, polycystic kidneys, hypopigmented hair, as well as abnormalities in development, bone formation and life span (Cell 75, 229). These symptoms are typically known as deficiency of kidneys in traditional Chinese medicine. We hypothesize that the Chinese concept of the Kidneys is related to the in vivo roles of the Bcl-2 family and the traditional Chinese herbal formulates designed for affecting the functions of the Kidneys could modulate the expression of this gene family. To test this hypothesis, the blood were collected from the volunteers before and after their taking the Jin Gui Shen Qi Wan (Jin Gui) Pills for Tonifying Kidney-energy, a classic herbal formulates believed to affect functions of the Kidneys. The levels of Bcl-2, Bax, Bcl-x_L and Bcl-x_s in lymphocytes were detected by flow cytometry. The collected serum from volunteers before and after the herbal treatment were added into in vitro culture systems of lymphocytes isolate from untreated volunteers. The apoptotic rates were quantified at 18 hours by flow cytometric analysis upon annexin V labeling. The results indicated that, after taking Jin Gui Shen Qi Wan, the fluorescing intensities of Bcl-2 increased while that of Bax decreased. The fluorescing intensities of both Bcl-x_L and x_s increased. The apoptotic rates in cultured lymphocytes were decreased in presence of the serum taken from volunteers after Jin Gui Shen Qi Wan use (n=9, P < 0.05). Our results indicated that the Jin Gui Shen Qi Wan could affect the expression of Bcl-2 members and the serum from volunteers treated with this formula had effects on natural apoptosis. We believe that this finding provides a useful way to identify effective compounds based on the effects on the expression the Bcl-2 family proteins and apoptosis. Additionally, our results also cast new light on understanding the unusual organ concept of ancient Chinese and integrating alternative medicine into the mainstream medicine.

Supported by the National Natural Science Fund of China (39900197) and Young Talent Grant of Scientific Bureau of Pudong New District, Shanghai (PK-99-13).

Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Beijing 100875, China
Tel: 010-62207368 E-mail: sangjianli@fm365.com

To study the cytotoxic effects and antitumour mechanism of norcantharidin(NCTD) on human breast cancer cell, MCF-7 cells were exposed to culture medium with NCTD in different doses and hours. The cell growth inhibition curves of NCTD on MCF-7 cells were acquired by detecting the growth ratio of MCF-7 cells with MTT methods. The results indicated that NCTD had inhibition effects on growth of MCF-7 cells depending on the doses and times of treatment. When the concentrations of NCTD used was lower than 5 mg/ml, the cells grew as same as the control cells. With increasing doses and times of treatment, the inhibition effects of NCTD on the cells' growth increased, for example, when exposed to 10 mg/ml NCTD for 24 h, the cell growth inhibition percentage was 39.40%, and exposed to 20 mg/ml, the percentage was up to 57.74%. But with the dose of NCTD increasing, the raise of the ratio of inhibition effects was not obvious.

For cells treated with 20mg/ml NCTD cells grew slowly in 24 h, then cells stopped growth and begen apoptosis.

NCTD had the effects of inhibition on the cell growth, but it was more important that NCTD can induce the apoptosis of MCF-7 cells. We extracted the DNA of several groups of cells treated by NCTD in different doses, and DNA ``Ladder" can be detected in all treated groups by using agarose gel electrophoresis. Analyzing the same samples by flow cytometry, the apoptosis peak can also be detected. Moreover, using two-photon laser microscopy and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, Fluo 3-AM, 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and rhodamine-123, it was found that the NCTD induced changes of nuclear morphology, reactive oxygen species (ROS), intracellular calcium concentration ([Ca²⁺]) and membrane potential of mitochondrion in MCF-7 cells. The results indicated that with the doses of NCTD increasing, the amount of apoptosis cells was climbing up, which had nuclear chromatin condensation, breaking-up marginalizing as well as apoptosis bodies. Compared to control cells, the cells that have characteristic apoptosis appearances had more high concentration of intracellular reactive oxygen species (ROS) and calcium concentration ([Ca²⁺]) but lower or none membrane potential of mitochondrion. It suggested that NCTD can induce the apoptosis of MCF-7 cells, and the intracellular reactive oxygen species (ROS), membrane potential of mitochondrion and calcium concentration([Ca²⁺]) might be involved in the induced apoptosis.

Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
E-mail: chris.bleackley@ualberta.ca

Cytotoxic T lymphocytes and natural killer cells protect us against a variety of pathogens, and are involved in autoimmune diseases and transplant rejection. Granzyme B plays a major role in the mechanism used by these cytolytic effectors to destroy the target cells. This proteinase is produced by the effectors and stored within cytoplasmic granules. Upon interaction with the pathogenic cell the granzyme is transferred from the CTL to the target via directed exocytosis and subsequent receptor mediated endocytosis. It is interesting that cells that are deficient in expression of the receptor for granzyme are also resistant to CTL-mediated killing. This represents an interesting target for the development of novel therapies for cancer and immunosuppression.

Once the granzyme is released within the target cell it can proteolytically activate a number of substrates that initiate apoptosis. Recent experiments have suggested that this is a multifaceted pathway that involves both direct and indirect action on caspases. The latter involves the cleavage of bid which subsequently translocates to mitochondria and promotes release of cytochrome c. However, we have also observed a direct induction of mitochondrial dysfunction by granzyme B. This suggests that additional substrates for the protease exist that may be involved in the death pathway when apoptosis is inhibited.

Akt and Bcl-x_L are independent regulators of the mitochondrial cell death pathways

Departments of Medicine and Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania, PA 19104, USA
E-mail: kerns@mail.med.upenn.edu

Akt and Bcl-x_L both promote resistance to apoptosis. A comparison of Akt- and Bcl-x_L-dependent cell survival was undertaken. Expression of constitutively active Akt allows cells to survive for prolonged periods in the absence of growth factors. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic gene Bcl-x_L. Although both genes prevent cell death, Akt-protected cells can be distinguished from Bcl-x_L- protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-x_L- expressing cells deprived of growth factor survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-x_L suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-x_L maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells.

The relevance of these in vitro observations to thein vivo regulation of cell survival has been examined. In vivo, lymphocytes require continuous signals from their microenvironment to prevent activation of the endogenous programmed cell death machinery. Thus, cell survival is limited in vivo by the availability of both ligands for receptors that directly or indirectly maintain cell survival. Lymphocytes deprived of receptor stimulation undergo progressive atrophy before commitment to apoptosis. Following loss of receptor engagement, lymphocytes rapidly downregulated the glucose transporter, Glut1. This was accompanied by reduction in mitochondrial potential and cellular ATP, suggesting that atrophy resulted from depletion of glucose-derived metabolic substrates. Concomitant with this nutrient deprivation, the lymphocytes adopt an anergic phenotype in which their ability to proliferate or secrete cytokines is severely compromised. Expression of Bcl-x_L prevented death but not the atrophy or unresponsiveness that follows either growth factor or glucose withdrawal. In Bcl-x_L transgenic animals, size, metabolic, and effector function of naive T cells were regulated through the T cell receptor (TCR) and correlated with TCR-dependent Glut1 expression. Akt has been implicated in both glycolytic regulation and cell size control. Like Bcl-x_L transgenics, Akt transgenics accumulate excess T cells. In contrast, however, Akt expressing cells are larger, maintain a higher mitochondrial potential, and are hyper-responsive. All of these functions are dependent on the availability of extracellular glucose and Akt promotes cell survival in response to growth factor but not glucose withdrawal. Together, these data suggest that cell survival and size control are regulated in response to extracellular signals which promote nutrient uptake and ATP production. Neither the cellular ATP/ADP ratio or mitochondrial potential appear to be regulated in a cell autonomous fashion in metazoan cells.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
E-mail: YShi@molbio.princeton.edu

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. We determined the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage in between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180-degree-flipping of the N-terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of induced fit. The inhibitor of apoptosis (IAP) proteins suppress cell death by inhibiting the catalytic activity of caspases. We determined the crystal structure of caspase-7 in complex with a potent inhibitory fragment from XIAP at 2.45 Å resolution. An 18-residue XIAP peptide binds the catalytic groove of caspase-7, making extensive contacts to the residues that are essential for its catalytic activity. Strikingly, despite a reversal of relative orientation, a subset of interactions between caspase-7 and XIAP closely resemble those between caspase-7 and its tetrapeptide inhibitor DEVD-CHO. Our biochemical and structural analyses reveal that the BIR domains are dispensable for the inhibition of caspase-3 and -7. This study provides a structural basis for the design of the next-generation caspase inhibitors.

¹Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115
²Current address: Division of biomaterials and tissue engineering, Biomedical engineering center, Industrial Technology Research Institute, Hsinchu, Taiwan 300

As one of the most characterized cytokines, interleukin 3 (IL-3) is well known for its survival effect on both progenitors and mature blood cells. Although with the extensive studies, the signaling pathways and underlying mechanism leading to survival responses of IL-3 still are not completely understood. Recently, an apoptotic genetic pathway of C. elegans was suggested to be evolutionally conserved in that controls the cytokine-dependent anti-apoptotic responses in mammalian hematopoietic cell lineages. Among this pathway, Ces-2 is known to be the first death specification gene in the C. elegans pathway and encodes a bZIP family transcriptional factor that shares the same DNA recognition sequence with an oncoprotein, E2A-HLF, created by chromosome translocation in certain human acute lymphoblastic leukemia. In the past several years, we devoted in the identification of cellular transcriptional factors that can recognize the CES-2/E2A-HLF binding element (CBE) and are involved in apoptotic regulation. We firstly demonstrated the existence of multiple binding complexes of CBE in various mammalian cell lines and tissues, and identify cAMP- responsive element binding protein (CREB) as a component in one major CBE-complex of Ba/F3 and TF-1 cells. Stimulation of Ba/F3 cells with IL-3 promptly induced phosphorylation of CREB at serine133 partially via a PKA-dependent pathway and activation of CREB resulted in induction of the CBE-driven reporter gene. Alteration of function of PKA or CREB strongly correlates with the survival capacity of Ba/F3 cells. Subsequently, we explored the IL-3-dependent transactivation mechanism of another CBE-binding protein, E4BP4. E4bp4 , a member of CBE-binding bZIP transcriptional factor subfamily, has been shown to be up-regulated by the IL-3 and plays an important role in IL-3's anti- apoptotic response. In this study, we demonstrated that E4bp4 is regulated by IL-3 mainly at the transcriptional level. Promoter mapping and binding analyses revealed that GATA-1 and GATA-2 proteins bind to a critical GATA site in the E4bp4 promoter in vitro and the chromatin immunoprecipitation (ChIP) assay further confirmed the in vivo binding of GATA-1 to the E4bp4 promoter. Overexpression of GATA factors transactivates the E4bp4 reporter, and alteration of binding levels of GATA-1 to the GATA site not only modulates the expression of the E4bp4 gene but also influences apoptosis induced by IL-3 removal. In summary, our works clearly demonstrated that these transcriptional factors are involved in transducing the IL-3 survival signal and their expression and function are both critical to IL-3's survival response. The discovery of GATA factor functioning in the upstream of the E4bp4 gene also extends our knowledge on the apoptotic pathway in hematopoietic cells, and may contribute to our further understanding of the processes of hematopoiesis and leukemogenesis.

Selection and study on minoligand of Acanthamoeba} which could induce tumor cells to apoptosis by phage display technique

¹ Life Science College, East China Normal University, Shanghai 200062, China
² Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
³CSIRO, Australia Animal Health Laboratory, Geelong VIC 3220, Australia

Acanthamoeba are free-living protozoa organisms that live abroad in nature. Pathogenic Acanthamoeba can cause a fatal granulomatous amoebic encephalitis and keratitis. Some species of Acanthamoeba can induce some tumor cells apoptosis in vitro. From 1996 we have proved that Acanthamoeba L strain (A. lugdunensis-A. quina), firstly got by us from keratitis patients in China, could induce tumor cells' apoptosis, including PC12, B16 cells et al. In this research we proved that both the body and the lysis of Acanthamoeba could induce mouse melanoma B16 cells apoptosis. But the apoptosis mechanism was poorly understood. According to our study, Acanthamoeba could band with the receptors on the tumor cells' surface, which would start the progress of tumor cells apoptosis. So, the 15-peptide library was screened to get some targeted peptides that could mimic Acanthamoeba material.

First, the random 15-peptide library was panned twice by intact B16 cells and then the special phages were panned by Acanthamoeba L strain antibody. After screening, many colonies specific were got by dot-ELISA technique. Finally 8 targeted phage peptides were obtained whose affinity are more than others do among those peptides by an advanced immunohistochemical method. These targeted phage peptides were sequenced and analyzed. The Western-blotting of phages also was proved that it was on PIII protein where they banded. To identify the specificity of targeted phage peptides, immunofluorescence technique was used to identify the binding of phage and cancer cells. Western-blotting data also showed that the targeted peptides could bind with some B16 membrane protein which molecular weight is from 15KD-43KD. To prove whether these targeted peptides could induce cancer cells apoptosis, Wright dyeing and other methods were used to detect the changes of B16. These phages did have some effect on the cells. This result was also proved by tetrazolium dye-based assay (MTT). The effect of the targeted phages from the former experiments was more than that of wild-type phages and irrelevant phages. And the effect changed when different amount of anti-Acanthamoeba antibody was added since the antibody counteracted the effect of targeted phages. This research provides a new method to identify the structure and also be likely to lead to the development of research on the pathogenesis of Acanthamoeba.

Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL 60615, USA
E-mail: alin@huggins.bsd.uchicago.edu

The proinflammatory cytokine tumor necrosis factor-a (TNF-a) regulates immune responses, inflammation and programmed cell death. The ultimate fate of a cell exposed to TNF-a is determined by signal integration between its downstream effectors, including caspases, I kB kianse (IKK) and c-Jun N-terminal protein kinase (JNK). However, the molecular mechanisms are incompletely understood. We investigated this issue using genetic and biochemical approaches. We identified IKKb, a catalytic subunit of the IKK complex that is required for NF-kB activation and cell survival in response to TNF-a, was proteolyzed by casp-3-related caspases. This proteolysis eliminated IKK activity and promoted TNF-a, killing. Point mutations that abrogated IKKb proteolysis generated a caspase-resistant IKKb mutant, which suppressed TNF-a induced apoptosis. Thus, IKKb proteolysis by caspases is a critical turning point in TNF-a, induced apoptosis. We also discovered that in addition to inhibiting caspases, NF-kB activation prevented prolonged JNK activation by TNF-a. This negative regulation contributed to inhibition of TNF-a induced apoptosis. The mechanisms behind NF-kB-mediated inhibition of JNK activation and the contribution of JNK activation to TNF-a induced apoptosis will be discussed.

Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL-null mice.

S-E LAMHAMEDI-CHERRADI¹, Shi Jun ZHENG¹, Kimberly A MAGUSCHAK¹,
Jacques PESCHON², Youhai H CHEN¹

¹Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
² Immunex Corporation, Seattle, WA, USA
E-mail:yhc@mail.medupenn.edu

Immunological tolerance to self is essential for maintaining the integrity of the organ systems, and its breakdown may lead to the development of autoimmune diseases. Tolerance to self is maintained through several mechanisms, which include negative selection, functional inactivation (anergy) and suppression of autoreactive lymphocytes. However, only negative selection permanently removes autoreactive cells through apoptosis. While it has long been known that negative selection requires a T cell receptor (TCR) signal, it is unclear whether a death ligand signal is also involved. TRAIL, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand, is a newly described member of the TNF family. Unlike other death ligands of the TNF family, TRAIL selectively induces apoptosis of tumor cells, thymocytes and hepatocytes. The roles of TRAIL in health and disease are yet to be established. Using five different models of negative selection, we show here that T cell death in the thymus requires not only a T cell receptor signal but also a death receptor signal mediated by TRAIL. Mice deficient in TRAIL have defects in extrinsic and intrinsic pathways of thymocyte apoptosis, and are highly susceptible to autoimmune diseases. Thus, TRAIL mediates negative selection and prevents autoimmune inflammation.

Program in Cellular & Molecular Biology and Department of Veterinary & Animal Sciences, University of Massachusetts, Amherst, MA 01003
E-mail: osborne@vasci.umass.edu

We have identified several key events in thymocyte apoptosis over the past few years, including a required role for proteasome function and the production of ROS. While we our data established that proteasome function and ROS production are required for apoptosis in thymocytes, we had not established the order of events leading from the primary apoptotic stimulus to the activation of caspases. Recently, we have demonstrated that both T cell receptor induced apoptosis and glucocorticoid induced apoptosis signal thymocytes to die through activation of the proteasome and this event is upstream of the production of ROS. Furthermore we have identified ROS production to occur within the mitochondrial membrane, most likely at complex III in the electron transport chain. ROS production is required for apoptosis and we show that it leads to cytochrome C release from the mitochondria and subsequent membrane depolarization. These events lead to the activation of the apoptosome and caspase 3 activation. We will present data demonstrating the requirement for these events as well as the order of these critical features in the commitment of thymocytes to apoptosis.

Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of apoptosis in T helper subset differentiation

Xiao Ren ZHANG¹, Satish DEVADAS², Li Ying ZHANG^1,2, Li LI¹, Achsah D KEEGAN¹, Yu Fang SHI^1,2

¹Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855;
²Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
E-mail: shiyu@umdnj.edu

Upon activation, naive T-helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of co-stimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate differentiation program, the selective action of death effectors determines the endpoint balance of differentiating T helper subsets. We examined the expression of TNF-related apoptosis-inducing ligand (TRAIL) and CD95L in cloned and in vitro differentiated Th1 and Th2 cells. We found that activation-induced expression of TRAIL is exclusively observed in Th2 clones and primary T helper cells differentiated under the Th2 condition, while the expression of CD95L is mainly in Th1 cells.

Furthermore, these two subsets exhibit distinct susceptibilities to TRAIL and CD95L mediated apoptosis. Th2 cells are more resistant to either TRAIL- or CD95L-induced apoptosis than Th1 cells. More importantly, both Th1 and Th2 cells could induce apoptosis in labeled Th1 but not Th2 cells. When AICD was examined, Th1 cells exhibited a Fas-FasL dependent caspase dependent apoptotic pathway and were rescued by a FasL antibody, ZVAD and TR6. Th1 cells were not rescued by DR5 or by OPG. Th2 cells also died upon restimulation but could not be rescued by Th1 anti apoptotic agents. Blocking TRAIL and CD95L significantly enhances IFN-g production in vitro. Likewise, young MRL/MpJ-lpr/lpr mice also showed more Th1 response to ovalbumin immunization as compared to MRL/MpJ-+/+. Therefore, apoptosis mediated by CD95L and TRAIL is critical in determining the fate of differentiating T helper cells.

Departments of Medicine and Microbiology, Boston University School of Medicine, and Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston, MA 02118, USA
E-mail: trothstein@MEDICINE.BUMC.bu.edu

The susceptibility of primary B cells to Fas (APO-1, CD95)-mediated apoptosis is regulated by signals derived from additional surface receptors. CD40 engagement produces upregulation of Fas expression and induces marked sensitivity to Fas-induced cell death, whereas B cell antigen receptor (BCR) engagement inhibits Fas killing and thereby produces Fas-resistance, even in otherwise susceptible, CD40-stimulated targets. BCR signaling for inducible Fas-resistance develops over a period of 12 hours and depends on new gene expression and new protein synthesis. The pathway from BCR to Fas-resistance bypasses Btk, but requires PKC and, notably, NF-kB. Although CD40 engagement triggers nuclear NF-kB expression, subsequent addition of anti-Ig further activates NF-kB. Inhibition of NF-kB activation at the time of anti-Ig addition blocks induction of Fas-resistance and this is true for chemical inhibitors such as lactacystin and PDTC, as well as for an IKBa dominant negative TAT fusion protein inhibitor. Several genes have now been implicated as proximal mediators of Fas-resistance in B cells, including NF-kB -responsive genes encoding FLIP and Bcl-x_L, and Fas Apoptosis Inhibitory Molecule (FAIM). The latter is a novel anti-apoptosis transcript identified by differential display that is broadly expressed and highly evolutionarily conserved. An alternative, longer form of faim has been identified that is highly restricted in its tissue distribution. The organization of the faim locus on murine chromosome 9F1 (human chromosome 3q21-25) indicates that the short and long forms of faim/FAIM arise through alternative splicing. This genomic information has been used to construct a FAIM knock-out animal that is viable but whose immunological characteristics have not yet been determined. The modulation of B cell susceptibility to Fas killing is likely to be important in normal and abnormal immune responses. Surface immunoglobulin-induced resistance to Fas-mediated apoptosis may protect antigen-specific B cells during potentially dangerous interactions with T cells, whereas aberrant acquisition of Fas-resistance may allow autoreactive B cells to escape deletion, leading to a breakdown of tolerance and an autoimmune phenotype.

Failure to disengage PI-3K pathway signaling confers Anti-IgM resistance to growth arrest and apoptosis in the CH12 B-cell lymphoma

Department of Immunology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA
E-mail: ScottD@usa.redcross.org

We and others have firmly established that surface IgM receptor (sIgM-R) crosslinking with antibodies to the i heavy chain (anti-i) leads to growth arrest and apoptosis in a series of well characterized B-cell lymphomas. This requires ablation of c-Myc protein expression and the concomitant induction of the cyclin-dependent-kinase inhibitor, p27^Kip1. The signaling mechanisms regulating c-Myc and p27^Kip1 protein expression are poorly understood. However, we recently established that sIgM-R mediated down-modulation of the PI-3K pathway directly affected c-Myc and p27^Kip1 expression and accurately predicted growth arrest or apoptosis in the ECH408 B lymphoma line. Moreover, p27-TAT fusion protein (which is rapidly taken up by these lymphoma lines) disappeared rapidly in growing B-lymphoma lines, but it persisted in the presence of anti-i . We have now extended these studies to the well-characterized CH12 B-cell lymphoma line, which is resistant to anti-i induced growth arrest and apoptosis, despite its high level expression of sIgM-R. The results show that failure to inactivate the PI3K effector, p70^S6K results in anti-i resistance. Blockade of PI-3K, or an upstream regulator regulator of p70^S6K, mTOR, reconstitutes reconstitutes or mimics all negative sIgM receptor signaling in these cells. Finally, anti-i or PI3K induce PTEN protein expression especially in the WEHI-231 B-cell line. Together, these results reveal apparently fundamental mechanisms for inducing immune tolerance (PI-3K suppression leading to anergy or deletion), or activation (stimulation of or maintenance of PI-3K activity), or lymphomagenesis (failure to disengage PI-3K signaling).

Transfusion of donor apoptotic splenocytes prolongs cardiac allograft survival in absence of immunosuppression

¹Organ Transplantation Department, Zhujiang Hospital, First Military Medical University, Guangzhou, China E-mail: ewsun@263.net
²Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

Although apoptosis has been recently documented to transmit immunosuppressive signals, their significance in allogeneic transplantation has not been reported. In the present study, we investigated the influence of donor apoptotic splenocytes on allograft survival in a SD to Wistar rat cardiac transplant model. Donor splenocytes were isolated and irradiated with ultraviolet (UV) to induce apoptosis in vitro. 5¡Á10⁷ apoptotic, necrotic or untreated donor spleen cells were transfused preoperatively and cardiac allogeneic transplantation was performed under no immunosuppression. The results showed that transfusion of donor apoptotic spleen cells preoperatively prolonged allograft survival without any immunosuppression on recipients. In contrast, recipients received necrotic or untreated donor spleen cells rejected the grafts at the same tempo as those transfused with saline. Administration of apoptotic cells at different time revealed that one week before transplantation operation was the most suitable time for graft prolongation. Splenocytes from recipients that have been transfused with donor apoptotic cells were co-cultured with irradiated donor splenocytes to test one-way mixed lymphocyte reaction (MLR). We found that splenocytes from recipients that have received donor apoptotic cells displayed a decreased proliferation in MLR. Grafts procured one week after transplantation showed no obvious histological rejection in recipients having received donor apoptotic spleen cells while strong rejection response found in the grafts from those that transfused with necrotic or untreated spleen cells. The results demonstrated that allogeneic graft prolongation could be achieved by donor apoptotic cells transfusion without system immunosuppression.

Peripheral clonal deletion of MBP-specific T cells by intravenous autoantigen: rapid reversal of ongoing, progressive experimental autoimmune encephalomyelitis

Guang Xian ZHANG¹, Divina CALIDA¹, Thomas T LIU², Elvia S VENTURA¹, You Hai CHEN², AM ROSTAMI¹

¹Department of Neurology, ²Institute for Human Gene Therapy, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
E-mail: gxzhang@mail.med.upenn.edu

Experimental autoimmune encephalomyelitis can be initiated spontaneously and developed progressively in TCR transgenic mice specific for myelin basic protein when exposed to non-sterile environment, thus more closely mimicking human multiple sclerosis. By intravenous administration of myelin basic protein, we succeeded in rapidly reversing the clinical and pathological signs of progressive spontaneous disease in these mice. The majority of transgenic T cells of MBP-injected mice was deleted, with dramatically increased numbers of apoptotic cells, in lymph nodes and spleen, but not in thymus. Proliferative responses of single transgenic T cell to autoantigen was significantly decreased in MBP-injected mice compared with PBS-injected mice, indicating that the MBP-specific T cells surviving from apoptosis were anergic. Moreover, production of both Th1 and Th2 cytokines was suppressed in MBP-injected mice. This study provides direct evidence that apoptosis-induced clonal deletion, along with anergy of autoreactive T cells surviving from spoptosis, play a major part in the rapid reversal of ongoing progressive EAE by intravenous administration of autoantigen.

Tullia LINDSTEN, Wei Xing ZONG, Jeffrey GOLDEN, Alan V WHITMORE, Marian HARRIS, Martin C RAFF, Craig B THOMPSON

Departments of Pathology and Laboratory Medicine, Cancer Biology and the Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA, and MRC Laboratory for Molecular Cell Biology, Cell Biology Unit and Biology Department, University College London, Gower Street, London WC1E 6BT, UK E-mail: lindsten@mail.med.upenn.edu

It is well recognized that cell death plays an important role during the maturation of the nervous system as well as in many neurological diseases. Apoptosis has been shown to be important particulary during embryogenesis as a means to eliminating unwanted neurons. Severed axons have also been shown to degenerate in an organized fashion termed Wallerian degeneration. Excitotoxic death is another form of cell death in the nervous system which is induced by high concentrations of neurotransmitters such as glutamate. It is not known whether the same molecular mechanisms underlie these different forms of cell death in the nervous system. The Bax-/-Bak-/- double knock-out mouse provides an ideal system to study the role of the pro-apoptotic Bcl-2 family members to answer some of these questions. Using this mouse model we have shown that Bax and Bak play an essential role in the regulation of normal tissue homeostasis as well as developmental apoptosis. In examining the brain of the Bax-/-Bak-/- mouse an accumulation of small neuronal cells with dense chromatin staining was observed. These cells were found in areas where during development neural progenitor cells are known to reside i.e. in the periventricular area, hippocampus, cerebellum and the olfactory bulb. These cell masses were also observed in areas where neural progenitor cells are normally not found as in the dorsal midline of the mesencephalon. Immunohistochemical analysis revealed that these cells were not of lymphocytic origin. Further analysis demonstrated that these cell masses contained a mix of cell types with staining positive for nestin (a neural progenitor cells marker), NeuN, NFP (neuronal markers) as well as for GFAP (astrocyte maker). In vivo Brdu labeling shows increased staining indicating that cell division is taking place. In order to study the effects of apoptotic stimuli neural progenitor cell cultures were established from brains of Bax-/-Bak-/- and control mice. Neural progenitor cultures from Bax-/-Bak-/- brains but not from wildtype brains were completely resistant to apoptosis induced by serum, N2 supplement and FGF removal, etoposide and staurosporine. The neural progenitor cell cultures from Bax-/-Bak-/- brains were shown to remain viable for as long as six weeks in culture following removal of serum and FGF. These cultures resumed proliferation after re-addition of growth factors. These results points to a role for Bax and Bak in death of neuronal cell bodies as illustrated both by the persistence of neuronal progenitor cells in the brains of double-knock-out mice and the resistance of neural progenitor cell cultures to a variety of apoptotic stimuli. We next studied axonal or Wallerian degeneration in the Bax-/-Bak-/- mice by severing the optic nerve from the retinal ganglion cell layer. Whereas the retinal ganglion cells remained viable the cut optic nerve displayed Wallerian degeneration similar to that found in normal mice. Thus, Wallerian degeneration is not dependent on Bax and Bak. Finally, we assessed the role of Bax and Bak in excitotoxic death using cerebellar ganglion cell cultures established from Bax-/-Bak-/- and normal mice. It was shown that Bax-/-Bak-/- neurons were just as susceptible to cell death induced by glutamate as cultures from normal mice. In contrast, treatment of the same Bax-/-Bak-/- cerebellar ganglion cell cultures with thapsigargin, showed complete resistance. These studies show that whereas Bax and Bak are essential for apoptosis of neurons, the mechanisms of axonal degeneration and exicitoxic death are not dependent on the activities of these two genes.

Laboratory of Gene Regulation and Development, NICHD/NIH, Bethesda, MD 20892, USA
E-mail: shi@helix.nih.gov

Anuran metamorphosis involves systematic transformations of individual organs in a thyroid hormone (TH)-dependent manner. Morphological and cellular studies have shown that the removal of larval organs/tissues such the tail and the tadpole intestinal epithelium is through programmed cell death or apoptosis. Recent molecular investigations suggest that TH regulates metamorphosis by regulating target gene expression through thyroid hormone receptors (TRs), which are DNA-binding transcription factors. Cloning and characterization of TH response genes show that diverse groups of early response genes are induced by TH. The products of these TH response genes are believed to directly or indirectly affect the expression and/or functions of cell death genes, leading to apoptosis of larval cells. Among the TH response genes isolated are a group encoding matrix metalloproteinases (MMPs). In vivo and in vitro studies suggest that extracellular matrix remodeling by MMPs plays an important role in regulating apoptosis and cell migration during tissue remodeling.

Institute of Hygiene and Environmental Medicine, Da-Li Road 1, Tianjin 300050, China
E-mail: qianlj2000@hotmail.com

Stress induced the serious disorder of cardiac function and cardiovascular diseases. Apoptosis is the cellular basis in stress induced cardiac injury. In our previous study we found that many stressors resulted in mitochondrial damage. It is certain that mitochondria is important mediator in triggering apoptotic cell death, but the mechanism, by which the stress induced mitochondrial injury leads to cardiomyocyte apoptosis, remains unclear. We designed the present study to investigate the changes of the mitochondria in cardiomyocytes undergoing stress and its role in inducing apoptosis. Here we reported that stress changed the membrane fluidity of mitochondria and induced the lipid peroxidation of mitochondrial membrane in cardiomyocyte in time and dose dependent fashion. The decrease of Bcl-2 expression and increase of Bax expression were observed on the mitochondria membrane in stressed cardiomyocytes. These stress induced alterations leaded to the opening of mitochondrial membrane permeability transition pore (MPTP). After mitochondrial MPTP opening there was a serious biological event happening, including disrupting of electron transport, uncoupling of oxidative phosphorylation, disorder of calcium metabolism, which in turn resulted in unbalance of intercellular homeostasis, characterized by alteration of cellular redox potential, calcium overload. It was more important that CytoC released from mitochondria to cytosol. Thus, caspase cascade was activated and apoptosis was initiated. By regulating the Bcl-2 overexpression in cardiomyocytes via gene transfection the alteration of mitochondrial membrane permeability transition was blocked and efflux of Cyto C form mitochondria was prevented. Fas mediated signal pathway is another important apoptotic mechanism. It was found that stress induced the increase of Fas expression on cardiomyocyte membrane, which activated caspase-8 and caspase-3 in succession. Hsp70 overexpression induced by heat preconditioning had no effect on Fas expression, but blocked caspase-8 activation and prevented partly the mitochondria mediated apoptosis. These finding implied the mitochondria as stress sensor and death signal integrator play a key role in initiating and mediating apoptosis of cardiomyocytes undergoing stress.

Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka, Suita Osaka 565-0871, Japan
E-mail: nagata@genetic.med.osaka-u.ac.jp

Apoptosis can be triggered by a variety of stimuli including death factors, anti-cancer drugs and factor-deprivation. These apoptotic cells are swiftly phagocytosed by macrophages to prevent the release of noxious or inflammatory materials from dying cells. The molecular analysis of Fas ligand (a death factor)-induced apoptosis indicated that a cascade of proteases (caspases) is activated during this process, which eventually activates a specific DNase (caspase-activated DNase). CAD exists as a complex with its inhibitor (ICAD) in proliferating cells. When the cells are triggered to apoptosis, caspases, in particular caspase 3, in the downstream of the caspase cascade cleave ICAD, which releases CAD to cause DNA degradation in nuclei. The cells deficient in the CAD system did not show any DNA fragmentation during apoptosis, indicating that the CAD system is solely responsible for the cell-autonomous apoptotic DNA fragmentation. However, the mice deficient in the CAD system developed normally, and TUNEL-positive cells were generated in situ as efficiently as the wild-type mice. A close examination of these apoptotic cells indicated that these cells are in phagocytes, suggesting a DNase (DNase II) in lysosomes of macrophages can digest the DNA of apoptotic cells non cell-autonomously. The mice lacking the DNase II gene died prenatally due to severe anemia. Macrophages in the fetal liver of the DNase II- null embryos carried many undigested nuclei, suggesting that the nuclei of erythroid cells are phagocytosed by macrophages during enucleation. If macrophages cannot digest the engulfed nuclear DNA, they may lose the ability to support erythropoiesis. To elucidate the molecular mechanism of phagocytosis of apoptotic cells, we established an assay system using CAD-deficient cells and peritoneal macrophages. Screening of several thousands monoclonal antibodies against the peritoneal macrophages identified a factor that links apoptotic cells to phagocytes. The factor was secreted by activated macrophages, specifically bound to apoptotic cells by recognizing phosphatidylserine, and stimulated non-pahgocytes to engulf the apoptotic cells.

MRC Human Immunology Unit, Institute of Molecular Medicine, University of Oxford, Headington, Oxford, UK
E-mail: xiaoning@molbiol.ox.ac.uk

Many viruses establish life-long infections in their natural host with few if any clinical manifestations. The relationship between virus and host is a dynamic process in which the virus has evolved the means to coexist by reducing its visibility, while the host immune system attempts to suppress and eliminate infection without damage to itself. We are now beginning to understand that viruses can employ a variety of strategies to evade host immune responses. These include escape from T cell recognition, resistance to apoptosis, and/or counterattack. Interestingly HIV which has a very short genome uses all of these strategies during the course of the infection.

A novel mediator of the amyloid-$\beta$ neurotoxicity in Alzheimer's disease: Ubiquitin conjugating enzyme E2-25K/Hip-2 as upstream of caspase-12

Yu Hyun SONG¹, So Young KIM¹, Yeon Mi HONG¹, Joo Yong LEE², Yung Joon YOO¹, Yong Keun JUNG^1*

¹Department of Life Science, Kwangju Institute of Science and Technology, Kwangju 500-712, Korea,
²University of Ulsan College of Medicine, Seoul 138-736, Korea
^*E-mail: ykjung@kjist.ac.kr

Accumulation of amyloid-b (Ab) and its neurotoxicity are regarded as a major factor promoting neuronal degeneration in Alzheimer's disease (AD). Upon investigation of Ab toxicity using DNA microarray, we isolated ubiquitin conjugating enzyme E2-25K/Hip-2 as a mediator of Ab toxicity. Here we show that expression of E2-25K/Hip-2 was strongly up-regulated in the cultured cortical neurons exposed to Ab _1-42 in vitro and in vulnerable neurons surrounding senile plaques of the brain derived from AD patients and Tg2576 Alzheimer's mice. Ab_1-42-induced neurotoxicity, accumulation of ubiquitin conjugates, and decrease of the proteasome activity were mediated by ubiquitin ligase activity of E2-25K/Hip-2. Ab -induced accumulation and activation of caspase-12 were coordinated by E2-25K/Hip-2 and dominant negative caspase-12 attenuated the E2-25K/Hip-2-induced cell death. These results suggest that E2-25K/Hip-2 is an important factor coordinating Ab toxicity in vulnerable neurons, leading to neuronal damage in the brain of AD patient.

Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Acute promyelocytic leukaemia (APL) has been attracting a wide interest far beyond hematological field in the last dozen years due to the presence of specific chromosome tranlocations and clinical responsibility to all-trans retinoic acid (ATRA) by differentiation induction as well as to arsenic trioxide (ATO). Most ( > 95%) APL patients carry specific chromosome translocation t(15;17), which leads to discovery of PML gene on chromosome 15. Such a translocation causes the fusion of PML to retinoic acid receptor-alpha (RARa) gene on chromosome 17 and expresses leukaemia-generating PML-RARa fusion protein.

PML has fascinated cell biologists because it localized to discrete nuclear subdomains known as PML nuclear bodies. These nuclear bodies are disrupted in leukemic cells that express PML/RARa in an ATRA or ATO -reversible mannerof the nuclear bodies, indicating that interference of PML/RARa with PML nuclear body formation could modulate is necessary for function of PML. Interestingly, alterations of PML expression have been observed in a variety of human tumours. What is the function of PML. As a candidate of transcription factors, wild-type PML was regarded as an important cell growth and apoptosis regulator. PML-null mice were shown to be highly sensitive to carcinogens and resistant to a variety of apoptotic signals. Conversely, PML overexpression induces apoptosis, growth arrest and senescence possibly mediated by transcriptional activation of p53.

Recently, ATO was worldwide confirmed to effectively treat APL, which was initiated in China. Thus, mechanisms of actions of ATO become a widely studied field. In vivo and in vitro data suggested that induction of apoptosis and partial differentiation are likely to constitute the cellular basis of the effects of ATO to treat APL. Here, mechanisms of ATO-induced apoptosis will be discussed. Briefly, ATO-induced apoptosis is not limited to APL cells. Actually, pharmacological concentrations of ATO also inhibit cell growth and/or induce apoptosis among some lymphomas and solid tumor cell lines in a dose-dependent way. On the other hand, mitochondria disorder and caspase activation are involved in ATO-induced apoptosis. More interestingly, ATO was also shown to modulate and degrade PML and PML/RARa proteins. In addition, ATO as a pleiotropic agent may have additional actions that also contribute to the induction of cell death, such as the activation of c-Jun NH2-terminal kinases and inactivation of the GTP binding site of monomeric tubulin.

Activation of p53 in gastric cancer cells by tripchlorolide specifically induces apoptosis

Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Research Center of Life Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
E-mail: wujr@sunm.shcnc.ac.cn

There are two possible outcomes when DNA damage occurs in normal mammalian cells: either induction of cell-cycle checkpoint which inhibits the progress of the cell cycles as well as activates DNA repair pathways, or activation of apoptosis to eliminate damaged cells. The p53 tumour-suppressor gene plays a key role in selecting these pathways. In our present works, the human gastric cancer cell line AGS was treated with tripchlorolide, a potent antitumor compound purified from a Chinese herb Tripterygium Wilfordii Hook. Single cell gel electrophoresis (Comet assay) showed that the treatment of tripchlorolide resulted in DNA damage in AGS cells. The damaged AGS cells went through apoptosis, which was time- and dose- dependent. Further analysis showed that this reagent caused the up-regulation of p53 both in RNA and protein levels, and the phosphorylation of p53 at Ser15. Interestingly, we cannot detect the increase of p21waf1/cip1, neither at the transcription-level nor at the protein-level, suggesting that activation of p53 does not up-regulate the transcription of p21. In order to confirm this phenomenon, we analyzed the in vivo DNA-binding ability of p53 using chromatin immunoprecipitation. The results showed that the drug-activated p53 barely bound to the promoter region of p21waf1/cip1. In addition, we established a p53 dominant-negative cell line AER by selecting an AGS clone stably expressing dominant-negative p53. Comparing to p53+/+ AGS, AER showed the resistance to the tripchlorolide-treatment. These results support the conclusion that tripchlorolide causes the DNA damage of AGS cells and activates p53, while the activation of p53 results in apoptosis of AGS cells rather than inhibition of AGS cell cycle.

Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati Medical Center, Cincinnati, OH 45267-0521
E-mail: XUMN@UCMAIL.UC.EDU

Apoptosis is a highly regulated physiological process critical in development and tissue homeostasis. Abnormal apoptosis can lead to disease conditions including neurodegeneration, autoimmunity and cancer. DNA fragmentation is an integral part of apoptosis and has long been suspected to be of critical importance in cleaning up potentially antigenic DNA and genetic material capable of inducing neoplasmic transformation in neighboring cells. Direct evidence for this function of DNA fragmentation however, is still lacking. The identification of a heterodimeric DNA fragmentation factor 45 and 40 (DFF45 and DFF40, also called ICAD for Inhibitor of Caspase Activated DNase and CAD for Caspase Activated DNase respectively) as well as endonuclease G (EndoG) provides a timely opportunity for addressing the significance of DNA fragmentation in apoptosis and tissue homeostasis. We previously generated a DFF45 mutant mouse in which the DFF activity is abolished. We found that DFF45-deficient thymocytes are resistant to DNA fragmentation both in vivo and in cultured primary cells exposed to various apoptotic stimuli. Interestingly, DFF45-deficient thymocytes and mouse embryonic fibroblasts (MEFs) are partially resistant to apoptosis in response to several apoptotic-inducing agents. DFF45 mutant mice also exhibit reduced natural killer T cell death after lymphocytic choriomeningitis virus infection. Furthermore, DFF45 mutant mice exhibit more granule cells in the dentate gyrus of the hippocampal formation compared to those in normal control mice. This increased neuronal cell number correlates with enhanced spatial and non-spatial learning and memory retention in DFF45 mutant mice compared with control mice. These results suggest that DFF45 is critical for DNA fragmentation and a deficiency in DFF45 can affect timely completion of apoptosis and consequently tissue homeostasis and proper cellular function. Likely due to the unaffected EndoG activity however, residual DNA fragmentation can be found in DFF45-deficient splenocytes and MEFs. In a collaborative effort, we are generating EndoG mutant mice and mice with combined deficiencies of DFF45 and EndoG to investigate how DFF and EndoG jointly function to insure proper apoptosis and tissue homeostasis.

Cloning and identifying of a novel protein binding with death domain of the death receptor 4

National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, PUMC and CAMS, Beijing 100005, China
E-mail: zheng@public3.bta.net.cn

DR4 (Death Receptor 4) belongs to the tumor necrosis factor (TNF) receptor gene family, which is defined by similar, cysteine-rich extracellular domain and a homologous cytoplasmic sequence termed as ``death domain". DR4 can transmit apoptosis signal initiated by Apo2L/TRAIL (TNF-related apoptosis inducing ligand). It can activate caspases within seconds of ligand binding and cause an apoptotic demise of the cell within hours. Despite several investigations, the mechanisms of apoptosis initiation by Apo2L/TRAIL remain unclear.

To investigate the mechanism of apoptosis signaling mediated by DR4, the yeast two- hybrid system was used for cloning and identifying proteins binding with the death domain of DR4. In brief, the death-domain of DR4 (DR4DD) was used as the bait to screen the human leukocyte MATCHMAKER cDNA library. Sequence and functional domain analysis was carried out by using automatic sequencing, BLAST and ScanProsite softwares, respectively. Two positive clones, named as pADB1 and pADB2, were obtained. Sequencing analysis showed that the insert fragments in pADB1 and pADB2 contained open reading frames fused with the GAL4AD sequence. pADB1 contained an open reading frame of 504 bp encoding 168 amino acids. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of human formyl peptide receptor-like 1 (FPRL1) was 97%. The insert of pADB2 shared no homologous with any known sequences in Genebank. The open reading frame of pADB2 was 141 bp encoding 47 amino acids. FPRL1 binds specifically with DR4CD (the cytoplasmic domain of DR4) in vitro and in vivo were confirmed by pull-down experiment and co-immunoprecipitation, suggesting that FPRL1 binds to DR4CD specifically.

In order to study the function of FPRL1 in signal transduction of cell apoptosis initiated by Apo2L/TRAIL, the cDNA of FPRL1 was cloned into the eukaryotic expression vector of pcDNA6/V5-HisB-HA and transiently transfected HEK293T. After 24 hours, the cells transfected by FPRL1/pcDNA6/V5-HisB-HA showed lower sensitivity to Apo2L/TRAIL treatment compared with control, suggesting that FPRL1 participates in the signaling pathway and regulation of apoptosis in HEK293T cells mediated by DR4.

Regulation of the subcellular localization of TRAF2 by TRAF1 reveals mechanisms of TRAF2 signaling

Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
E-mail: ychoi3@mail.med.upenn.edu

TRAF2 is a critical adaptor molecule for TNF receptors in inflammatory and immune signaling. Upon receptor engagement, TRAF2 is recruited to CD40 and translocates to lipid rafts in a RING finger-dependent process, which enables the activation of downstream kinases. TRAF1 can displace TRAF2 and CD40 from raft fractions, and it promotes the ability of TRAF2 to sustain signal activation. Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cytoskeletal protein Filamin, but not NF-kB activation. TRAF1-/- dendritic cells show attenuated responses to secondary, antiapoptotic stimulation by TRAF2-dependent factors. These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of TRAF2 signaling including anti-apoptotic processes.

Characterization of a novel proapoptotic protein, map-1, that associates with bax through its multiple bcl-2 homology domains

Kuan Onn TAN, Shing Leng CHAN, Naiyang FU, Karen Mei Ling TAN, Karen SY YEE, Victor C YU

Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
E-mail: vckyu@atozasia.com

Members of Bcl-2 family of proteins are regulators of cell death that can be grouped into subfamilies of prosurvival and proapoptotic molecules. They are characterized by the presence of several conserved motifs, known as the Bcl-2 homology (BH) domains, designated BH1, BH2, BH3 and BH4. Mutagenesis and structural studies revealed that the BH domains are important functional domains that are also required for dimerization function. Recently, a subfamily of proapoptotic molecules only contains BH3 motif has been identified suggesting BH3 domain alone may be sufficient for mediating proapoptotic function among members of the proapoptotic subfamily in the BCL-2 family.

Interestingly, gene deletion analyses, however, revealed that cells lacking both Bax and Bak (two key members of the ``multidomain" proapoptotic Bcl-2 subfamily), but not cells lacking one of the two genes, are completely resistant to apoptotic death triggered by diverse stimuli. Thus, activation of a ``multidomain" proapoptotic member, BAX or BAK, appears to be essential for apoptotic signaling.

To gain further understanding the molecular functions of the ``multidomain" proapoptotic molecules, we used yeast two hybrid screen to identify protein partners of BAX. A novel proapoptotic protein, termed MAP-1, was identified as a BAX-interacting protein from a human brain cDNA library. MAP-1 contains a BH3-like motif and mediates caspase- dependent death in mammalian cells when overexpressed. MAP-1 homodimerizes and associates with the proapoptotic BAX and the prosurvival Bcl-2 and Bcl-XL of the Bcl-2 family in vitro and in vivo in mammalian cells. Mutagenesis analyses revealed that the BH3- like domain in MAP-1 is not required for its association with Bcl-XL, but is required for association with BAX and for mediating apoptosis. Interestingly, in contrast to other BAX- associated proteins, the binding of MAP-1 to BAX appears to require all three BH domains (BH1, BH2 and BH3) of BAX.

RC DUKE^1,2, K HANCE¹, J SUN¹, D CHAN², SJ MEECH³, B PIETRA², D NELSON⁴, D BELLGRAU^1,2

¹ GlobeImmune, Inc., Denver, Colorado,
² University of Colorado School of Medicine, Denver, Colorado
³ Brown University School of Medicine, Providence, Rhode Island
⁴ Children's Hospital Medical Center, Cincinnati, Ohio

Fas ligand (FasL) was first described functionally as an inducible cell surface molecule used by cytotoxic T cells to induce apoptotic cell death in tumor cells and activated lymphocytes. With the identification of Fas as the lpr gene product, FasL became recognized as a molecule involved in down-regulation of the immune system. While FasL can be used to efficiently kill Fas-expressing tumor cells as well as activated T and B lymphocytes in vitro, attempts to use FasL therapeutically to treat cancer or to prevent transplant rejection have not been successful due to unexpected toxicity. In this talk, we will present results using mutated versions of FasL in the settings of cancer gene therapy, arthritis and heart transplant.

This work was supported in part by USPHS-NIH SBIR grants AI-40394, AI-40607, and AI-47168.

Cloning and identification of apoptosis specific DNase inhibitor IXAD in Xenopus egg extract

College of Life Sciences, Peking University, Beijing 100871, China
E-mail: 1. zhglu@pubms.pku.edu.cn; 2. zhangcm@pku.edu.cn; 3. zhaizh@plum.lsc.pku.edu.cn

XAD is an apoptosis specific DNase in Xenopus and can cut the linker-DNA between nucleosomes during apoptosis in Xenopus laevis egg extract induced by cytochrome c. XAD is most likely to be the homologue to DFF40 in humans. The activity of DFF40 can be inhibited by DFF45. We report molecular cloning and identification of an XAD inhibitor, IXAD (inhibitor of XAD), in Xenopus eggs. We cloned the complete cDNA of the DFF45 homologue, IXAD. There is a specific DEVD sequence in its N-terminal, which serves as the cleavage site of XCPP32 (the caspase 3 in Xenopus). IXAD is abundant in Xenopus egg extract and exists either as dimmer or as a complex with XAD. During apoptosis, IXAD is degraded and its DNase inhibitory effect was eliminated. Based on these results, we conclude that IXAD is an apoptosis specific DNase inhibitor in Xenopus and can inhibit apoptosis through inhibiting XAD.

N-Myc downstream-regulated gene 2 (NDRG2) is a negative regulator of cell growth and its expression is decreased in cancers

Li Bo YAO, Yan Chun DENG, Shu Jun LI, Ling CHAU, Wo Shing AU, Xin Ping LIU, Ji Cun WANG, Jian LI, Fu Yang LI, Shao ping JI, Hsiang Fu KUNG, Suet Yi LEUNG, Marie CM LIN

Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi'an, China;
Institute of Molecular Biology and Open laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong, China
E-mail: bioyao@fmmu.edu.cn

Using subtraction cloning, we identified the human N-Myc Downstream- Regulated Gene-2 (hNDRG2), located at 14q11.2, as a candidate tumor suppressor gene. Semi-quantitative RT-PCR showed that the expression of hNDRG2 in 15 of 27 (56%) human GBM tissues and all 6 human glioblastoma cell lines was significantly lower than that in the normal brain. The expression of hNDRG2 also was evaluated in 60 lung- carcinoma patients. 17 of 26 cases of squamous carcinoma and 4 of 11 cases of small cell lung cancer displayed the low expression of hNDRG2. In addition, the down- regulation of hNDRG2 was observed in hypatoma and leukemia. Consistently, transfection of human glioblastoma U373 cells with hNDRG2 markedly reduced the cell proliferation. Human lung carcinoma cell line GLC-82 transfected with hNDRG2 also demonstrated the cell growth inhibition and G1 arrest. Our findings provide the first evidence to suggest that hNDRG2 may play an important role in carcinogenesis.

Selective induction of apoptosis in K562 cells by a BCR-ABL tyrosine kinase inhibitor, STI571

Medical Research Center of Nanjing Drum Tower Hospital, Nanjing University, Nanjing 210008, China
E-mail: keyanbu33@hotmail.cim

To study the mechanism and specificity of anti-proliferative effect of STI571 on BCR- ABL positive cell line. BCR-ABL positive human leukemia cell line K562 and BCR- ABL negative human leukemia cell line MO7E were employed. Cells were treated with STI 571 or Adriamycin for 18 hours and their morphology and genomic DNA integrity were investigated. STI 571 induced apoptosis in K562 cells but not in MO7E cells. In contrast, adriamycin induced apoptosis in MO7E cells but not in K562 cells. STI 571 shows anti-proliferative effects on BCR-ABL positive cells by blocking the activity of BCR-ABL tyrosine Kinase and induces apoptosis. It has little inhibitory effects on BCR- ABL negative cells. We concluded here that the inhibitory effect of SRI571 is specific on the BCR-ABL signal transduction pathway.

The protective role of heat shock protein against cardiomyocyte apoptosis by mitochondrial pathway

Wei Min XIAO, Bi Mei JIANG, Yong Zhong SHI, Mei Dong LIU, Dao Lin TANG, Xian Zhong XIAO

Department Of Pathophysiology, Xiangya Medical College, Central-South University, Changsha 410078, China
E-mail: xianzhongxiao@hotmail.com

Apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements. Previous studies have shown that cardiomyocyte can be protected from ischemia-reperfusion (I/R) injury by heat shock proteins (HSP). However, to date the protective roles of HSP against cardiomyocyte apoptosis has not been confirmed .The present study was designed to explore the effects of mitochondrial pathway in the protective role of heat shock protein against cardiomyocyte apoptosis Cardiomyocyte apoptosis was induced by in vivo mouse heart ischemia-reperfusion (I/R) injury and by hydrogen peroxide (H₂O₂, 0.5 mM) in cultured neonatal rat cardiomyocyte and C₂C₁₂ cell line. (2) Cardilmyocyte apoptosis was determined by Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling (TUNEL), flow cytometric analysis, Hoechst 33258. (3)The activity of caspase 3,8,9 was assayed by Caspase Colorimetric Assay Kit (R&D)and Western-blot. (4)The distribution of cytochrome C and Smac in mitochondria or cytoplasm was observed by Indirect immunofluorescence and Westerm-blot. (1)Heat Shock response(42 ^oC,1 h) could increase HSP70 expression in mouse heart, cultured neonatal rat cardiomyocyte and C₂C₁₂ cell line. (2) The activity of Caspaes 3, 8, 9 increased after 30 minutes of coronary occlusion followed by 8 hours reperfusion in mouse heart,which could be inhibited by heat shock response. (3) Heat shock response could inhibit H₂O₂-induced cardiomyocyt apoptosis and activation of caspase 3, 8, 9 in cultured neonatal rat cardiomyocyte and C₂C₁₂ cell, (4) Heat shock response could inhibit H₂O₂ induced- translocation of cytochrome c and Smac from mitochondria to cytoplasm. The protective role of HSP70 against cardiomyocyte apoptosis induced by ischemia- reperfusion injury and H₂O₂ involved mitochondria pathway , in which HSP70 inhibited cytochrome C and Smac release from mitochondria and decrease activation of Caspase 9.

College of Life Sciences, Peking University, Beijing 100871, China
E-mail: 1, < greensky@pubms.pku.edu.cn > ; 2, < zhaizh@plum.lsc.pku.edu.cn > ; 3, < hongbingshu@hotmail.com >

AIF is a mitochondrial flavoprotein that triggers caspase-independent apoptosis. We cloned and characterized a novel AIF homologous molecule designated as AMID (AIF-homologous Mitochondrion-associated Inducer of Death. AMID lacks a mitochondrial localization sequence but shares significant homology with AIF and NADH-oxidoreductases from bacteria to mammalian species. Immunofluorescent staining and biochemical experiments indicated that AMID was co-localized with mitochondria. Overexpression of AMID induced cell death with characteristic apoptotic morphology. Furthermore, AMID-induced apoptosis was independent of caspase activation and p53, and was not inhibited by Bcl2. These findings suggest that AMID induces a novel caspase- independent apoptotic pathway.

15-deoxy-¦¤^12,14-prostaglandin-J₂ induces apoptosis in ECV304 endothelial cells by inhibiting NF-kB and AP-1 activation pathways

Department of Cardiology, First Affiliated Hospital, Zhongshan University, Guangzhou 510089, China

Prostaglandin J₂ (PG J₂) and its metabolites are naturally occurring derivatives of Prostaglandin D₂ (PG D₂). The pathway for formation of these compounds involves sequential conversion of PG D₂ to PG J₂, and 15-deoxy-\triangle^12,14-prostaglandin-J₂ (15d-PGJ₂). 15d-PGJ₂ is a high-affinity ligand for peroxisome proliferation-activated receptor gamma (PPARg), it represses several genes in different cell lines. Our previous studies have demonstrated that 15d-PGJ₂ induced apoptosis in ECV304 endothelial cells. However, the mechanism remains unclear. In this paper, our aim was to explore the molecular mechanism of 15d-PGJ₂ on apoptosis in ECV304 endothelial cells. Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), ELISA, flow cytometry, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to clarify the effect of 15d-PGJ₂ on apoptosis in ECV304 cells and related molecular mechanisms. The results showed that 15d-PGJ₂ (5-20 mM) induced apoptosis in a dose-dependent manner. Western blot analysis showed 15d-PGJ₂ inhibited the degradation of I kBa and the expression of c-jun protein. EMSA analysis confirmed that 15d-PGJ₂ strongly inhibited the DNA binding activity of nuclear NF- kB, and AP-1 in ECV304 endothelial cells. These results suggest that 15d-PGJ₂ induce apoptosis of ECV304 endothelial cells, which may be through inhibiting NF- kB, and AP-1 activation pathways.

How Bax sequesters its hydrophobic membrane anchor to regulate both subcellular localization and dimer formation

Bax, a pro-apoptotic member of the Bcl-2 family, changes intracellular location as it accelerates cell death. Bax consists of 9 a-helices where the assembly of helices a1-a8 resembles that of the anti-apoptotic protein, Bcl-x_L. The opposite biological functions between Bcl-x_L and Bax stem from relatively minor differences in their structures. The C-terminal a helix, that functions in mitochondrial membrane targeting, sits in the hydrophobic BH3 binding pocket proposed previously to mediate heterodimer formation among Bcl-2 family members. The structure of soluble Bax shows that the conformation of the C-terminal helix may simultaneously inhibit BH3 peptide binding associated with dimer formation and mitochondrial membrane insertion, a crucial early step in apoptosis. The binding of Bax to Bcl-x_L is increased in the presence of certain nonionic detergents. Using coimmunoprecipitation analysis we found that Bcl-x_L lacking the C-terminal hydrophobic tail fails to heterodimerize with Bax. Furthermore, Bax lacking its C-terminal tail binds Bcl-x_L even in the absence of detergents. These results suggest that Bcl-x_L binds via its C-terminal hydrophobic tail to the hydrophobic pocket in Bax in vitro. The topology of Bax and Bcl-x_L in mitochondrial membranes during apoptosis remains to be established.

Phosphoinositides as ligands of PHD-fingers: implications for chromatin remodeling

Or GOZANI¹, Philip KARUMAN¹, Seth FIELD¹, Hong ZHU¹, James CHA¹, Jennifer
VILLASENOR¹, Vikram R RAO¹, Joan S BRUGGE¹, Lewis C CANTLEY¹, Jun Ying
YUAN^1*, Cheryl L BAIRD², Bharat MEHROTRA², Jian CHEN², David G MYSZKA²,
Colin G FERGUSON², Glenn D PRESTWICH²

¹Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
²Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA
^*E-mail: Junying_-Yuan@hms.harvard.edu

Phosphatidylinositol phosphates (PtdInsPs) play critical roles in signal transduction pathways, cytoskeletal regulation, and vesicular trafficking and are implicated in multiple disease pathways. In the nucleus, PtdInsPs have been found associated with chromatin, but their specific nuclear-binding proteins have not been identified. In a biochemical screen for PtdInsP-binding proteins, we identified a protein (ING2) containing a Plant Homeodomain (PHD) finger, a motif present in many chromatin- regulatory proteins. We show that the PHD domains of ING2 and of other diverse nuclear proteins bind to the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we provide evidence for a functional interaction between the PHD domain of ING2 and phosphoinositides in vivo. Finally, we demonstrate that the PHD domain of ING2 regulates the association of p53 with chromatin. Our data identify the PHD-finger as a novel phosphoinositide-binding module, making it the first known nuclear PtdInsP-binding domain, and suggest that PHD-PtdIns(5)P interactions can directly regulate chromatin-remodeling processes.

Transforming growth factor-b1 suppresses serum deprivation-induced apoptosis via activation of ERK1/ERK2 and the inhibition of p38 activity and ceramide production

Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
E-mail: songj@sumn.shcnc.ac.cn

In this report we studied the effects and mechanism of transforming growth factor- b1 (TGF-b1) on serum deprivation-induced cell apoptosis. Serum deprivation induces apoptosis, which is associated with an increase in intracellular ceramide level and with the activation of p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 MAP kinase by SB203580 significantly reduced apoptosis induced by serum-deprivation. Treatment of cells with TGF-b1 stimulated cell proliferation and suppressed the serum deprivation-induced apoptotic response. The anti-apoptotic effect of TGF- b1 is correlated with its ability to inhibit the serum deprivation-induced activation of p38 MAP kinase and the increase in intracellular ceramide level. In addition, serum deprivation increases the SMase activity and a corresponding decrease in the level of sphingomyelin (SM), which can also be completely suppressed by TGF-b1. Over-expression of p38 MAP kinase increases the cell apoptosis and reduces the anti-apoptotic effect of TGF- b1. Furthermore, TGF-b1 also activates the ERK1/ERK2 MAP kinases. Treatment of cells with PD98059, a selective inhibitor of ERK1/ERK2, completely inhibited the TGF-b1-stimulated proliferation and partially inhibited the anti-apoptotic effects of TGF-b1. Moreover, inhibition of p38 MAP kinase by SB203580 also leads to suppression of apoptosis but not increased ceramide generation induced by serum deprivation. Similarly, PD98059 has no effect on TGF-b1-mediated inhibition of the serum deprivation-induced ceramide generation. Our data demonstrate that ceramide, p38 and ERK1/ERK2 MAP kinases played critical but differential roles in the survival, proliferation, and the stress-induced apoptosis in fibroblast cells. TGF-b1 suppresses the serum deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 and increased ceramide generation.

Activation/Inactivation of ion channels and pumps during apoptosis: a necessary role in the apoptotic volume decrease

John A CIDLOWSKI, Carl D BORTNER, Maria-Gomez ANGELATS, Alyson SCOLTOCK, Nina STOREY, David ARMSTRONG, Mona RAZIK

National Institutes of Environmental Health Sciences, NIH Research Triangle Park, NC 27705, USA
E-mail: cidlows1@niehs.nih.gov

During apoptosis cells undergo a series of evolutionarily conserved biochemical and morphological changes that include the loss of cell volume or an apoptotic volume decrease (AVD). This AVD response distinguishes apoptosis from other forms of cell death such as necrosis. Experiments in our laboratory and others have shown that AVD is both an early and necessary component of apoptotic death. We have now investigated the molecular basis for the AVD response in Jurkat cells and Hepatoma cells induced to undergo apoptosis with a variety of death stimuli. Our data reveal significant alterations in the flux of both sodium and potassium ions control the apoptotic AVD response. We will provide new data that show that K⁺ efflux is mandatory for both generation of AVD and the activation of apoptosis. Using both pharmacological probes and single cell patch clamp techniques we have now also identified some of the ion channels that are activated/inactivated during the AVD response. Based on these data we will show that ion channel inhibitors can be used to effectively block apoptosis and maintain long tern cell viability.

Xue Jun ZHANG^1*, Qi Huang JIN¹ , HY HE¹ , H JIANG¹ , Lei YANG¹ , ZQ JIANG¹ , YF CHAI¹ , Yu Fang SHI² ,

¹Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031, China;
²Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 661 Hoes Lane, Piscataway, New Jersey, NJ 08854, USA
^*E-mail: zhang-xj@sunm.shcnc.ac.cn

Acetylcholinesterase (AChE) plays a key role in terminating neurotransmission at cholinergic synapses. AChE is also found in tissues devoid of cholinergic responses, indicating its potential function beyond neurotransmission. It has been suggested that AChE may participate in development, differentiation, and pathogenic processes such as Alzheimer's disease and tumorigenesis. We examined AChE expression in human lung fibroblast cell line (HLF) upon induction of apoptosis by UV or G418. AChE is induced in all apoptotic cells examined as determined by cytochemical staining, immunological analysis, affinity chromatography purification, and molecular cloning. The AChE protein was found in the cytoplasm at the initiation of apoptosis and then in the nucleus or apoptotic bodies upon commitment to cell death. Sequence analysis revealed that AChE expressed in apoptotic cells is identical to the synapse type AChE. Blocking the expression of AChE with antisense i