Nuclear factor kB (NF-kB) is a transcription factor that controls the expression of many cellular and viral genes. The p65 (RelA) subunit plays a critical role as a transcriptional activator and recent observations have highlighted its role in the control of apoptosis. Here we report that 53BP2, a protein previously identi®ed by interaction with wild type p53 and Bcl-2, also binds to p65 in a yeast two-hybrid system. This speci®c interaction was con®rmed by pull-down assay in vitro and by a mammalian two-hybrid assay in vivo. We observed that full-length 53BP2 fused to GFP had a punctate distribution in cytoplasm, predominantly in perinuclear region whereas the N-terminal 53BP2 localized in cytoplasm and C-terminal 53BP2 localized in the nucleus. Furthermore, we found that overexpression of GFP-53BP2 induced apoptosis in transiently transfected cells. Neither the N-terminal nor the Cterminal of 53BP2 fused to GFP induced cell death. Interestingly, co-transfection with a p65 expression plasmid signi®cantly inhibited 53BP2-induced cell death. The previous ®ndings that 53BP2 bound to p53 and Bcl-2 together with our present observations suggest that 53BP2 may play a central role in the regulation of apoptosis and cell growth.

CRM1 plays an important role in the nuclear export of cargo proteins bearing nuclear exporting signal sequences. Leptomycin B (LMB), a well-known CRM1 inhibitor, possesses strong antitumor properties. However, its toxicity prevents it from being clinically useful. In this study, we demonstrate that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export, causing arrest of the cell cycle and inducing apoptosis in a time-and dose-dependent manner for

Although a major effect of p21, a cyclin-dependent kinase inhibitor, is considered to be exerted during G 1 phase of the cell cycle, p21 gene knock-out studies suggested its involvement in G 2 /M checkpoint as well. Here we demonstrate evidence that p21 is required for the cell cycle arrest at G 2 upon DNA damage. We found that expression of wild-type p21 (p21 WT ), not mutant p21 (p21 PCNA؊ ) lacking the interaction with proliferating cell nuclear antigen (PCNA), caused G 2 cell cycle arrest in p53-deficient DLD1 colon cancer cell line after the DNA damage by treatment with cis-diamminedichloroplatinum (II). We also found that p21 WT was associated with Cdc2/cyclin B1 together with PCNA. Furthermore, coimmunoprecipitation experiments revealed that PCNA interacted with Cdc25C at the G 2 /M transition, and this interaction was abolished when p21WT was expressed presumably due to the competition between p21 WT and Cdc25C in the binding to PCNA. These findings suggest that p21 plays a regulatory role in the maintenance of cell cycle arrest at G 2 by blocking the interaction of Cdc25C with PCNA.

Transcription from the human immunodeficiency virus type 1 (HIV-1) provirus is activated by a cellular factor, NF kappa B, recognizing the tandemly repeated 10-base-pair sequences, termed the kappa B sequence, present in the enhancer region within the viral long terminal repeat (LTR). Using electrophoretic mobility shift assay (EMSA), which demonstrates specific DNA-protein interaction in vitro, we could demonstrate that reducto-oxidative modulation of NF kappa B dramatically changes its DNA binding activity and that a cellular physiological reducing catalyst, thioredoxin (TRX) also known as adult T cell leukemia derived factor (ADF), fully restored the DNA-binding activity of the oxidized NF kappa B. We also observed that purified TRX/ADF protein could augment gene expression from HIV LTR as demonstrated by transient chloramphenicol acetyltransferase (CAT) assay. These observations confirmed the previous notion that ADF might be an inducing factor of cellular interleukin-2 receptor alpha subunit (IL-2R alpha) through the kappa B sequence that is a common central cis-regulatory element in both IL-2R alpha and HIV gene expression. These observations indicate that reducto-oxidative regulation (or redox regulation) of a cysteine residue(s) on the NF kappa B molecule might play an important role in its specific DNA interaction and that it might provide a clue to the understanding of a pathway of cellular signal transduction to NF kappa B that is independent from the known pathways involving protein phosphorylation.

We have purified the human lymphocyte Fc receptor specific for IgE (Fcc receptor) and its soluble form by using the anti-Fcc receptor monoclonal antibody H107. Using an oligonucleotide probe corresponding to the partial amino acid sequence of the soluble Fce receptor related to IgE binding factor, we cloned, sequenced, and expressed a cDNA for the receptor. The Fcc receptor has 321 amino acid residues with no NH2-terminal signal sequence. The receptor was separated into two domains by a putative 24-amino acid residue transmembrane region located near the NH2-terminal end. The Fce receptor showed a marked homology with animal lectins including human and rat asialoglycoprotein receptors, chicken hepatic lectin, and rat mannose binding proteins. Tresyl-Sepharose (Pharmacia) at 5 mg/1 ml of beads (10). Soluble FCE receptor was purified from the conditioned medium of RPMI 8866 cells using H107-Sepharose after absorption with the human gamma globulinSepharose. The FcE receptor was purified as described (8). The cell lysate of RPMI 8866 cells in 0.5% Nonidet P-40 was absorbed with bovine serum albumin-, human gamma globulin-, and DNase I-conjugated Sepharose, and then incubated with the H107-Sepharose. After extensive washing with a high-salt buffer (10 mM sodium phosphate, pH 7.5/0.5 M NaCl/0.1% Nonidet P-40), soluble FCE receptor and FcE receptors were eluted from H107-Sepharose with an elution buffer (0.1 M acetic acid, pH 4.0/0.5 M NaCl/0.1% Nonidet P-40). Affinity-purified, soluble FCE receptor was further fractionated on Superose column with Fast Protein Liquid Chromatography (FPLC; Pharmacia), to elucidate its IgE binding activity. NaDodSO4/PAGE and Protein Amino Acid Sequence. The method of NaDodSO4/PAGE is described elsewhere (8). For immunoblots, nonreducing 13% polyacrylamide gels were used. After electrotransfer to a nitrocellulose filter, the membrane, blocked with 3% (wt/vol) bovine serum albumin, was incubated with or without H107 mAb (1 ,g/ml) and then with goat anti-mouse IgG [F(ab')2] conjugated to horseradish peroxidase (Tago). The antibody-bound bands were visualized with 3,3'-diaminobenzidine tetrahydrochloride and H202. For amino acid sequencing, both affinity-purified, soluble FcE receptor and FcE receptor were further purified by reverse-phase HPLC (using a Synchropak RP-P C18 column) (Beckman) with a linear gradient of 2-propanol with Abbreviations: FC£ receptor, Fc receptor for IgE; IgE-BF, IgE binding factor; mAb, monoclonal antibody. tTo whom reprint requests should be addressed.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells. In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identi®ed proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C. The interaction between Cdc25C and PCNA was con®rmed in vitro and in vivo. Coimmunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis. Immuno¯uorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase. Together with the previous observations of the interaction between various cdc/ cyclin and PCNA, our ®ndings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle.

Cell cycle G 2 checkpoint abrogation is an attractive strategy for sensitizing cancer cells to DNA-damaging anticancer agent without increasing adverse effects on normal cells. However, there is no single proven molecular target for this therapeutic approach. High-throughput screening for molecules inhibiting CHK1, a kinase that is essential for the G 2 checkpoint, has not yet yielded therapeutic G 2 checkpoint inhibitors, and the tumor suppressor phenotypes of ATM and CHK2 suggest they may not be ideal targets. Here, we optimized two G 2 checkpoint-abrogating peptides, TAT-S216 and TAT-

Many conventional anticancer treatments kill cells irrespective of whether they are normal or cancerous, so patients suffer from adverse side effects due to the loss of healthy cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G2 checkpoint during cell replication. Damaged DNA in humans is detected by sensor proteins (such as hHUS1, hRAD1, hRAD9, hRAD17, and hRAD26) that transmit a signal via ATR to CHK1, or by another sensor complex (that may include γH2AX, 53BP1, BRCA1, NBS1, hMRE11, and hRAD50), the signal of which is relayed by ATM to CHK2. Most of the damage signals originated by the sensor complexes for the G2 checkpoint are conducted to CDC25C, the activity of which is modulated by 14-3-3. There are also less extensively explored pathways involving p53, p38, PCNA, HDAC, PP2A, PLK1, WEE1, CDC25B, and CDC25A. This review will examine the available inhibitors of CHK1 (Staurosporin, UCN-01, Go6976, SB-218078, ICP-1, and CEP-3891), both CHK1 and CHK2 (TAT-S216A and debromohymenialdisine), CHK2 (CEP-6367), WEE1 (PD0166285), and PP2A (okadaic acid and fostriecin), as well as the unknown checkpoint inhibitors 13-hydroxy-15-ozoapathin and the isogranulatimides. Among these targets, CHK1 seems to be the most suitable target for therapeutic G2 abrogation to date, although an unexplored target such as 14-3-3 or the strategy of targeting multiple proteins at once may be of interest in the future.