Low Levels Of Bcl-2 Research Articles

Modulation of the oncogenic potential of β‐catenin by the subcellular distribution of plakoglobin

Plakoglobin (Pg) and beta-catenin are homologous proteins that function in cell-cell adhesion and signaling. The cadherin-associated form of these proteins mediates adhesion, whereas the cytosolic/nuclear form has a signaling role. Despite their interactions with common cellular partners, beta-catenin has a well-documented oncogenic potential while Pg has a less characterized tumor suppressor activity. We showed previously that Pg overexpression in Pg-deficient SCC9 cells (SCC9-Pg-WT) induced Bcl-2 expression and inhibited apoptosis. To assess the exact role of Pg in Bcl-2 expression, we generated and characterized SCC9 transfectants expressing Pg with a restricted cytoplasmic (Pg-NES) or nuclear (Pg-NLS) distribution. We show that Bcl-2 was expressed regardless of Pg localization, although its level was substantially lower in SCC9-Pg-NLS cells. Bcl-2 expression coincided with increased nuclear beta-catenin levels (Pg-NES) or a decrease in the level of total and nuclear beta-catenin associated with N-cadherin and alpha-catenin (Pg-WT and -NLS) cells. Bcl-2 expression also was induced in SCC9 cells overexpressing beta-catenin. In contrast, SCC9 cells expressing mutant Pg proteins, unable to interact with N-cadherin and alpha-catenin, had noticeably lower Bcl-2 levels. Our data suggest that Bcl-2 expression is induced by beta-catenin and modulated by Pg. We show that the inhibition of beta-catenin-dependent TCF transactivation had no effect on Bcl-2 levels, suggesting that induction of Bcl-2 expression by beta-catenin and its modulation by Pg may involve factors other than, or in addition, to, TCF. These results provide a possible mechanism for the tumor suppressor activity of Pg via its role as a regulator of the oncogenic potential beta-catenin.

Bcl-2 Does Not Rescue T Lymphopoiesis in Interleukin-7 Receptor Transgenic Mice (95.15)

Abstract We have previously demonstrated that murine thymocytes down-regulate IL-7 sensitivity between the β-selection and positive selection checkpoints. To assess the consequences of enforced IL-7Rα expression during this developmental phase, we evaluated thymocyte IL-7 responsiveness in transgenic mice expressing the IL-7Rα gene under control of the lck proximal promoter. This model demonstrated that induction of ectopic high-level IL-7Rα expression during thymopoiesis increases the percentage of thymocytes responsive to IL-7 but results in an overall decrease in total thymic cellularity in transgenic mice. Transgenic thymocytes initiated IL-7Rα expression beginning in the DN2 compartment which was maintained through development to naïve peripheral subsets. Interference with thymocyte development became apparent shortly after initiation of transgene expression as there was an ~65% reduction in DN3 thymocytes in transgenic mice. This finding was reminiscent of phenotypes observed in IL-7 signaling-deficient mice insofar as there was a significant loss of pre-T cells, which correlated with a reduction in Bcl-2 expression. Indeed, lower Bcl-2 levels were observed in transgenic DN2 and DN3 thymocytes when assessed directly ex vivo. Additionally, and unlike the IL-7 signaling-deficient models, transgenic restoration of Bcl-2 expression did not correct the thymic hypocellularity induced by IL-7Rα overexpression. These data demonstrate that ectopic overexpression of the IL-7Rα past the pro-T stage interferes with DN3 thymocyte development and inappropriately maintains IL-7 responsiveness in β-selected thymocytes.

Early apoptosis and cell death induced by ATX-S10Na (II)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (II). HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting. Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-x(L), were decreased in Bax- and p53-independent manner. Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na (II) are mediated by p53-Bax network and low levels of Bcl-2 and Bcl-x(L) proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.

Two overrepresented B cell populations in HIV-infected individuals undergo apoptosis by different mechanisms

Perturbations of B cells in HIV-infected individuals are associated with the overrepresentation of distinct B cell populations. Here we describe high extrinsic CD95 ligand (CD95L)-mediated apoptosis in CD10-/CD21lo mature/activated B cells that likely arise from HIV-induced immune activation. In addition, high intrinsic apoptosis was observed in CD10+ immature/transitional B cells that likely arise as a result of HIV-induced lymphopenia. CD10+ B cells expressed low levels of Bcl-2 and Bcl-xL, consistent with their high susceptibility to intrinsic apoptosis. Higher levels of activated Bax and Bak were induced in CD10+ B cells compared with CD95L-treated CD10- B cells, consistent with the greater involvement of mitochondria in intrinsic vs. extrinsic apoptosis. Of interest, both extrinsic apoptosis in CD95L-treated CD10- B cells and intrinsic apoptosis in CD10+ B cells were associated with caspase-8 activation. Our data suggest that two distinct mechanisms of apoptosis are associated with B cells of HIV-infected individuals, and both may contribute to the depletion and dysfunction of B cells in these individuals.

The role of skin-homing T cells in extrinsic atopic dermatitis

T cells that express Cutaneous Lymphocyte-Associated antigen (CLA) have the potential of migrating to the skin, and are hypothesized to play a role in cutaneous atopic disease. To investigate the immune phenotype and cytokine responses to Der p 1 stimulation of CLA+ T cells in extrinsic atopic dermatitis (EAD). In vitro testing, with controls. Peripheral blood mononuclear cells (PBMC) were obtained from EAD patients (n=27) and non-atopic healthy individuals (n=22). Phenotypic analysis of naive, CLA+ and non-CLA+ memory/effector CD4+ and CD8+ T cells used markers of cell activation, differentiation, adhesion, apoptosis and chemokine receptor expression. Cytokine responses in these cells were studied following Der p 1 stimulation. CLA+ T cells from EAD patients expressed significantly higher levels of CD25, HLA-DR, CD38, CD71, CXCR1, CXCR2 and lower levels of bcl2, CCR5, CCR7, CXCR3, and CD62L (p<0.05). In EAD patients, CLA+ T cells express increased levels of markers associated with activation, adhesion and apoptosis, show differences in the level of expression of differentiation markers and display a distinct chemokine receptor preference, compared with cells from healthy controls. These data suggest a significant role for CLA+ T cells in the pathogenesis of cutaneous atopic disease.

Concomitant Inhibition of MDM2 and Bcl-2 Protein Function Synergistically Induce Mitochondrial Apoptosis in AML

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance ofantiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acutemyeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus,functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects ofMdm2 inhibition. We here investigate the potential therapeutic utility of combinedtargeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors ofprotein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational changeand mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3ainduced p53-mediated apoptosis predominantly in S and G2/M cells, while cells in G1 were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G1 , the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser70 was absent in G1 but detectable in G2/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G1 cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.

Bcl-2, Mcl-1 and p53 Expression Confer Sensitivity to Bcl-2 Inhibitor ABT-737 in Multiple Myeloma.

Bcl-2 family of proteins confers resistance to chemotherapy in multiple myeloma (MM). Identification of BH3-mimetic drugs that inactivate pro-survival targets by interfering with the interaction of Bcl-2 proteins may therefore be therapeutically useful. A recent study identify ABT-737, a potent small-molecule inhibitor of anti-apoptotic proteins Bcl-2, Bcl-xL, and Bcl-w with an affinity 2–3 orders of magnitude more potent than any previously reported compounds (Nature 2005, 435:672–681). Here we show that ABT-737 triggers apoptosis in MM cells resistant to conventional therapies. The functional specificity of ABT-737 binding to Bcl-2 or Bcl-xL was confirmed by using the less active enantiomer of ABT-737, which had no significant effect on viability of cells, even at higher concentration. Washout experiments show that exposure of MM cells to ABT-737 for 3h is sufficient to cause an early and irreversible commitment to cell death. Given that ABT-737 binds Bcl-2, Bcl-xL, and Bcl-w with high affinity but has far lower affinity for Mcl-1, we asked whether the anti-MM activity of ABT-737 is regulated by the relative expression of Bcl-2, Bcl-xL, or Mcl-1. For these studies, we selected MM.1S (sensitive to ABT-737: IC50-2 μM) and OPM-1 (least sensitive to ABT-737; IC50: 10–15 μM) and examined the basal expression levels of Bcl-2, Bcl-xL, and Mcl-1. MM.1S cells, in contrast to OPM-1 cells, express high basal levels of both Bcl-2 and Bcl-xL, but low Mcl-1 protein; conversely, OPM-1 cells express high Mcl-1 and low Bcl-2 levels. Since ABT-737 cannot neutralize Mcl-1, it is likely that the high Mcl-1 and low Bcl-2/Bcl-xL expression profile in OPM-1 renders these cells less sensitive to ABT-737; whereas low Mcl-1 and high Bcl-2/Bcl-xL expression in MM.1S cells allows for efficient killing by ABT-737. Importantly, treatment of MM.1S MM cells with ABT-737 and proteasome inhibitor Bortezomib induces additive cytotoxicity (combination index = 1.0). The mechanism underlying additive anti-MM activity of ABT-737 with Bortezomib includes downregulation of Mcl-1 along with targeting Bcl-2. Our findings have clinical implications: Bortezomib is FDA approved for the treatment of MM, but prolonged treatment can be associated with toxicity (Chauhan et al. Cancer Cell 2005, 8:407–419); importantly, combining ABT-737 with Bortezomib would allow for the use of lower doses of Bortezomib. Another mechanism whereby MM cells evades the cytotoxic effects of chemotherapy is via p53 mutations. Bcl-2 is linked to p53-mediated signaling, and we therefore examined whether ABT-737 is able to overcome the tumorigenic effects conferred via p53 mutations. Co-precipitations experiments show that MM.1S cells (ABT-737-sensitive) predominantly carry wild type p53, whereas OPM-1 cell line (less sensitive to ABT-737) has mutant p53. Taken together, these finding suggest that 1) sensitivity to ABT-737 correlates with Bcl-2 expression in MM cells, 2) higher expression of Mcl-1 reduces sensitivity to ABT-737 whereas other anti-MM agents that block Mcl-1 may synergize with ABT-737, and 3) ABT-737 induces apoptosis in MM with p53 (wt) or p53 (mt), albeit with differential sensitivity. A report that ABT-737 enhances the apoptotic activity of chemotherapeutic agents (Olterdorf et al. Nature 2005, 435:672–681), together with our present findings, provides the framework for clinical trials of ABT-737, either alone or in combination with other anti-MM agents, to enhance efficacy, reduce toxicity, and overcome drug resistance in MM patients.

Role of the Cyclic AMP Response Element in the bcl-2 Promoter in the Regulation of Endogenous Bcl-2 Expression and Apoptosis in Murine B Cells

We have previously shown for B-cell lines that the cyclic AMP response element (CRE) is a major positive regulatory site in the bcl-2 promoter. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutated CRE was introduced into the bcl-2 promoter region (mutCRE-bcl2 mice). Quantitative chromatin immunoprecipitation assays revealed that mutation of the CRE abolished the binding of CREB/ATF and CBP transcription factors to the bcl-2 promoter and greatly diminished the binding of NF-kappaB factors. The mutant CRE significantly reduced the expression of Bcl-2 in B cells and rendered them susceptible to surface immunoglobulin- and chemotherapeutic agent-induced apoptosis. The low levels of Bcl-2 were not changed with activation of the cells. The numbers of pre-B, immature B, and mature B cells in the bone marrow were decreased, as were the numbers of splenic B cells in mutCRE-bcl2 mice. Our findings indicate that the CRE in the bcl-2 promoter has an important functional role in the regulation of endogenous Bcl-2 expression and plays a critical role in the coordination of signals that regulate B-cell survival.

SPC2996—A Bcl-2 RNA antagonist being studied in chronic lymphocytic leukemia

6610 Background: Bcl-2 protein is a critical regulator of programmed cell death and contributes to apoptosis resistance in many cancers, including chronic lymphocytic leukemia (CLL). The novel RNA antagonist, SPC2996, is a 16-mer oligonucleotide incorporating Locked Nucleic Acid (LNA) with unique high-affinity binding to Bcl-2 mRNA and enhanced resistance to nuclease digestion. We report the pharmacological properties of this new investigational drug and are currently evaluating clinical safety and initial efficacy in CLL patients. Methods: In vitro, the effects of SPC2996 on Bcl-2 mRNA and protein levels and phenotypic responses were evaluated in human cancer cell lines by quantitative polymerase chain reaction, Western blotting and biochemical assays. In vivo bio-distribution in rodents was assessed with tritium-labelled SPC2996 given intravenously. Anti-tumour efficacy was assessed in human prostate (PC3) and melanoma (518A2 and 607B) tumour cell line xenografts in immunodeficient mice. Preclinical toxicology and safety pharmacology were evaluated after intravenous treatment in rats and primates and tissue uptake measured by HPLC. Results: In vitro, low nanomolar concentrations of SPC2996 substantially reduced intracellular levels of Bcl-2 mRNA and protein and induced apoptosis in a dose-dependant manner. In vivo, systemically administered SPC2996 accumulated in liver, kidney, bone marrow, and lymph nodes. Repeated dosing at 10mg/kg inhibited growth of human xenografts in mice. Substantial down-regulation of Bcl-2 mRNA was observed in primate tissues after intravenous administration of SPC2996 at 3 and 6mg/kg. No acute toxicity was encountered on systemic bolus dosing of substantially higher doses in primates. An international phase I/II dose-escalating study in 42 patients with relapsed or refractory CLL is currently recruiting in the USA, UK, Denmark and France. Conclusions: SPC2996 is the first member of a new generation of specific RNA antagonising oligonucleotides with enhanced properties derived from LNA. SPC2996 has the potential to lower Bcl-2 levels in CLL cells without significant toxicity and with less frequent dosing than required with previous drugs. [Table: see text]

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Effect of Bax G125A Single Nucleotide Polymorphism (SNP) on Bax Gene Expression in Diffuse Large Cell Lymphoma.

Altered expression of Bax protein has been shown to be predictive of overall and disease free-survival of patients with diffuse large B-cell lymphoma (DLCL)(Am J Hematol , 2003; 73:101–7). Further, absence of Bax and low levels of Bcl-2 have been shown to be associated with a more aggressive phenotype (P<0.05) (Blood , 1997; 90:3173–8) in patients with DLCL. We have previously reported a single nucleotide polymorphim (SNP), G125A, in the 5′-untranslated region of the BAX promoter and its association with reduced protein expression and treatment resistance in B-cell chronic lymphocytic leukemia (Cancer Lett 2002; 187:199–205). The aim of the present study was to evaluate BAX SNP incidence and its association with the protein expression in lymphomas. DNA was extracted from fifty lymphoma samples (31 DLCL, seven follicular lymphomas (FL), three marginal zone lymphomas (MZL), four T-cell lymphomas, three Hodgkin's lymphomas, and two cases of Mantle cell lymphoma (MCL), and from 55 control samples. BAX SNP was detected by restriction enzyme analysis (J Clin Pathol , 2003; 56: 205–9). Fourteen out of 50 (28%) samples had G125A SNP, in all cases only one allele was replaced. It was present in eight (26%) DLCL, two (29%) FL, in two MZL, in one T-cell lymphoma, in one HL, and none of MCL had this SNP. Eight out of 55 (14.5%) controls had the SNP. The presence of BAX promoter SNP was associated with reduced Bax protein expression as detected by Western blot analysis (P=0.024). Two plasmids with G and A nucleotides at position 125 of the BAX promoter linked to luciferase as a reporter were constructed and used for transfection, as well as the pGL3-Basic and pGL3-Control plasmids; pRL-SV40 plasmid was used for co-transfection. Dual-Glo luciferase assay was performed and Firefly luciferase activity was normalized to Renilla luciferase. In transfection experiments, the G125A SNP reduced expression (P=0.0202) of BAX promoter. BAX SNP, G125A, present in approximately a quarter of DLCL has a direct negative effect on BAX gene expression.

Bcl-2 Antisense Oligonucleotide Overcomes Resistance to E1A Gene Therapy in a Low HER2-Expressing Ovarian Cancer Xenograft Model

We are currently conducting clinical trials of E1A gene therapy for patients with ovarian cancer. The adenovirus type 5 E1A gene suppresses growth of ovarian cancer cells that overexpress HER-2/neu (HER2) and growth of some--but not all--that express low HER2. In HER2-overexpressing cells, suppression by E1A is predominantly by down-regulation of HER2, but the mechanism in low HER2-expressing cells is not fully understood. The adenoviral E1B protein has sequential and functional homology to Bcl-2 and prolongs the viability of adenovirus host cells by inhibiting E1A-induced apoptosis. Bcl-2 is overexpressed in ovarian cancer and participates in chemoresistance; we hypothesized that Bcl-2 inhibits E1A-induced apoptosis leading to resistance to E1A gene therapy. E1A suppressed colony formation of ovarian cancer cells that express low levels of Bcl-2 and HER2 (OVCAR-3 and OVCA 433), but enhanced colony formation in low HER2-, high Bcl-2-expressing ovarian cancer cells (2774 and HEY). Treating 2774 or HEY cells with antisense oligonucleotide Bcl-2 (Bcl-2-ASO) did not reduce cell viability. E1A combined with Bcl-2-ASO led to significant decreases in cell viability resulting from increased apoptosis relative to cells treated with E1A alone (P < 0.05). The increase in apoptosis was partly due to cytochrome c release and subsequently caspase-9 activation by Bcl-2-ASO. Finally, in an ovarian cancer xenograft model, treatment with Bcl-2-ASO did not prolong survival, but E1A plus Bcl-2-ASO did (P < 0.001). In conclusion, ovarian tumors overexpressing Bcl-2 may not respond well to E1A gene therapy, but treatment with a combination of E1A and Bcl-2-ASO may overcome this resistance.

Bcl-2 Positively Regulates Sox9-dependent Chondrocyte Gene Expression by Suppressing the MEK-ERK1/2 Signaling Pathway

Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase C alpha and NFkappaB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype.

Developmental Program of Epileptogenesis in the Brain of EL Mice

We recently observed inducible nitric oxide synthetase (iNOS) expression and decreased Cu, Zn-superoxide dismutase (Cu, Zn-SOD) activities in the hippocampus of epileptic mutant EL mice at the age of 30 weeks. In addition, the immediate early gene (IEG) c-fos is unusually expressed in the interictal period, suggesting activation of protein cascades associated with the epileptogenesis. Furthermore, DNA fragmentation has been detected preferentially in the hippocampus CA1 and the parietal cortex of EL mouse brain. It remains to be seen, however, how these abnormalities are related to the DNA fragmentation, and whether neuronal cell loss is involved. The present study was designed to address these issues. NOS isoenzymes, pro- (Bax) and antiapoptotic factors (Bcl-2, Bcl-XL), and neurotrophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; fibroblast growth factor-2, FGF-2) were determined by immunoblotting in the EL mouse brain at various developmental stages. Hematoxylin-eosin staining was applied to the formalin-fixed brains to examine the cell loss in the tissue. IEG expression in the interictal period was analyzed by in situ hybridization by using the 35S x-ray emulsion method. nNOS was the major component of NOS in the hippocampus of either EL or control DDY mice. In EL mice, however, iNOS was detectable at the age of 10 weeks, at which the animals usually experience the first seizures. eNOS, which appears in DDY brain, could scarcely be identified. Even in the interictal period, EL mice expressed c-fos continuously, preferentially in the parietal cortex and hippocampal CA1. In DDY mice, very low steady-state levels of Bcl-2 and Bax remained constant throughout development. In EL mice, these Bcl-2 and Bax levels were increased even before experiencing frequent seizures. BDNF in EL mice markedly increased temporarily during ictogenesis and epileptogenesis in their early periods. Unexpectedly, no cell loss was found in the hippocampus. DNA fragmentation without cell loss found in EL mouse brains appears to result from initial activation and later inactivation of the apoptotic process. Neurotrophic factors may play a role in the ictogenesis and the epileptogenesis during the early development. These gene expressions closely related to the periods critical for ictogenesis and epileptogenesis may be of particular importance in the development of antiepileptic drugs (AEDs) with novel mechanisms.

Effects of Low Dose Radiation on Tumor Apoptosis, Cell Cycle and Apoptosis-Related Protein Bcl-2 in Tumor-Bearing Mice

Objective: To study the effects of low dose radiation (LDR) on tumor apoptosis, cell cycle progression and changes of apoptosis-related protein Bcl-2 in tumor-bearing mice. Methods: Male mice of Kunming strain were implanted subcutaneously with S180 sarcoma cells in the left inguen as an in situ experimental animal model. Seven days later, the mice were subjected to 75 mGy whole-body γ-irradiation. At 24 and 48 h after the irradiation, all mice were sacrificed. The tumor sizes were measured, and tumor cell apoptosis and cell cycle progression were analyzed by flow cytometry. The expression of apoptosis-related protein Bcl-2 and the apoptotic rate of tumor cells were observed by immunohistochemistry and electron microscopy. Results: Tumors grew significantly slower after LDR (P<0.05). The tumor cells were arrested in G1 phrase and the expression of Bcl-2 protein decreased at 24 h. Apoptotic rate of tumor cells was increased significantly at 48 h after LDR (P<0.01). Conclusion: LDR could cause a G1-phase arrest and increase the apoptosis of tumor cells through the low level of apoptosis-related protein bcl-2 in the tumor-bearing mice. The organized immune function and anti-tumor ability are markedly increased after LDR. Our study provides practical evidence of clinical application to cancer treatment.

Oral treatment of the TRAMP mice with doxazosin suppresses prostate tumor growth and metastasis

We used the TRAMP mouse model for testing the effect of oral doxazosin treatment on the in vivo prostate tumor growth and metastasis. Five groups of TRAMP mice at different ages were orally fed with 1 mg/kg of doxazosin or DMSO for 45-196 days. At the end of oral treatment, tumor weight was determined, and metastasis to multiple organs examined. The levels of MUC18, Bcl-2, Bax, caspase-3, poly (ADP-ribose) polymerase (PARP), phospho (Ser473)-AKT, and Ki-67 in the mouse prostate tumors were determined. Oral treatment of the TRAMP mice with doxazosin for 45-81 days did not decrease the size of preexisting prostate tumors, but it limited the metastasis to peri-aortic lymph nodes. A prolonged treatment of TRAMP mice with doxazosin (156-196 days), if administered early, decreased the prostate tumor weight and completely suppressed metastasis. The doxazosin treatment did not further decrease the expression of an already low level of Bcl-2 in all prostate tumors, but it increased the expression of Bax, and the activation of caspase-3, and the cleavage of a downstream substrate, PARP. The treatment reduced the expression of MUC18, phospho (Ser473)-AKT, and Ki-67. The treatment in the early phase appeared to promote prostate tumor growth and increased the expression of a proliferative index, Ki-67. Doxazosin, if administered early, may be useful for preventing the prostate tumor formation, and also for limiting or completely suppressing the metastasis of prostate cancer in the TRAMP model. The mechanism of doxazosin is consistent with the established hypothesis.

Neuoscience: Molecular Pathology and Epilepsy

Abstract Yoshiya L. Murashima*, Jiro Suzuki*, and Mitsunobu Yoshii* *Department of Neural Plasticity, Tokyo Institute of Psychiatry, Tokyo, Japan . Purpose: We recently observed inducible nitric oxide synthase (iNOS) expression and decreased Cu, Zn-superoxide dismutase (Cu,Zn-SOD) activities in the hippocampus of epileptic mutant EL mice at age 30 weeks. In addition, DNA fragmentation was detected preferentially in region CA1 of the hippocampus and in the parietal cortex of the EL mouse brain. The results suggest that abnormalities in the region-specific iNOS isoenzyme activities and decreased Cu, Zn-SOD activities might produce spatially specific free radicals, leading to DNA fragmentation. However, neuronal cell loss is not found in the hippocampus. Furthermore, the immediate early gene (IEG), c-fos, also is expressed in the interictal period at age 30 weeks. These findings suggest that some protein cascades occur during epileptogenesis. In the present study, we used epileptic mutant EL mice to examine whether the altered equilibrium of the proapoptotic Bax and anti-apoptotic Bcl-2/Bcl-XL induces DNA fragmentation without cell loss. We also examined whether neurotrophic factors modulate the developmental process of epileptogenesis in EL mouse brain. Methods: Developmental changes of NOS isoenzymes were determined by using immunoblotting. Formalin-fixed mouse brains were stained by hematoxylin–eosin to examine cell loss in the tissue. IEG expression in the interictal period was analyzed with in situ hybridization by using the 35S x-ray emulsion method. Developmental changes of Bcl-2, Bcl-XL, and Bax were examined by immunoblotting in the hippocampus of EL mice compared with the control DDY mice. Alterations of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and fibroblast growth factor-2 (FGF-2) during development were investigated by immunoblotting. Results: Unexpectedly, the eNOS content in EL mouse brain was very low although eNOS appears to be responsible for NO that mediates an increase in local cerebral blood flow during focal seizures. Neuronal NOS, iNOS, and relatively lower concentration eNOS are essential to establish either ictogenesis or epileptogenesis. No cell loss was found in the hippocampus. Even in the interictal period, EL mice expressed the IEG, c-fos, continuously, preferentially in the parietal cortex and hippocampal CA1 when the seizure thresholds were very low. In the control DDY mice, very low steady-state levels of Bcl-2 and Bax were observed throughout development. In EL mice, the Bcl-2 and Bax levels increased predominantly after frequent seizures. The BDNF level in EL mice showed a significant increase during development, in the period of ictogenesis and epileptogenesis. Conclusions: The results indicate that in EL mice, the susceptibility of hippocampal neurons to DNA fragmentation increases after experiencing repetitive seizures during development. This effect probably is due to a change in the balance between the protective mechanism and inactivation of the proapoptotic pathway. BDNF may play a role in the ictogenesis and epileptogenesis by promoting abnormal synaptic plasticity.

Critical Molecular Studies to Strategically Plan Therapy in Chronic Lymphocytic Leukemia (CLL): Correlating Cellular Proteins with Defined Prognostic Patient Subsets and Their Response to Treatment.

The critical importance of specific proteins including Bcl-2, Mcl-1, XIAP, Bax, Caspase-3, Traf-1, and ZAP70 in predicting disease progression, prognosis, and response to therapy in CLL have been extensively studied through in vitro experiments. We now report molecular data obtained from a large prospective clinical trial involving 278 previously untreated patients with CLL. This ECOG-led intergroup trial involved participation from CALGB and SWOG, and currently provides quantitative information regarding protein levels on 217 cases assessed immediately prior to treatment. NCI criteria for therapy in CLL were met before patients were randomized to receive either Fludarabine or Fludarabine plus Cytoxan. On-study analyses included: interphase cytogenetics by Fluorescence In Situ Hybridization (FISH), p53 mutational analysis, immunoglobulin variable heavy chain (IgVH) gene rearrangement, and quantitative analysis of the aforementioned specific proteins. Peripheral blood mononuclear cells were obtained from each patient at registration before starting therapy. Lysates were prepared from the patient cells and separated by SDS-PAGE. Proteins were detected by immunoblot analysis using the multiple antigen detection method previously developed by our group. Detection was performed by an enhanced chemiluminescence method and quantified by densitometry. Expression levels were calculated relative to expression in the Jurkat T cell line (for ZAP70) or the RS11846 lymphoblastoid cell line (for apoptosis-related proteins). Using a two-sided Wilcoxon rank sum test, molecular data were compared with FISH and IgVH mutational status. Patients with either p53 mutations or del(17p) had significantly lower levels of XIAP (p=0.022). Patients with del(11q) had significantly higher Bcl-2/Bax ratios (p=0.052). Patients with trisomy 12 had lower levels of Bcl-2 (p=0.066); higher Mcl-1 (p=0.026); higher Bax (p=0.09); and higher Caspase-3 (p=0.019) levels. Interestingly, these specimens also had lower ratios of Bcl-2/Bax (p=0.0016). Patients with del(13q) had significantly higher levels of Bcl-2 (p=0.0059) lower levels of Caspase-3 (p=0.047), and higher Bcl-2/Bax ratios (p=0.0569). IgVH mutated patients had significantly lower levels of Caspase-3 using either a 98% cut-off (p=0.0008) or the 97% cut-off (p&lt;0.0001). Finally, patients with higher ZAP70 protein expression had significantly higher levels of Caspase-3 (p=0.0099) and Traf-1 (p=0.016). In 163 patients evaluable for both response and the molecular studies, either elevation of Bcl-2 protein (p= 0.01) or an abnormality of p53 (either del 17p or a p53 mutation) (p=0.08) correlated significantly with attainment of a complete remission. Potential opportunities to predict response to therapy in CLL will hopefully identify those patients likely to benefit from therapy. Based upon our previous work, these molecular parameters can be modulated both in vitro and in vivo. Therefore, understanding the relationship of these important proteins to therapeutic response can facilitate strategic plans to circumvent drug resistance to chemotherapy and immunotherapy in CLL.

Quantitative PCR Detection of t(14;18) bcl-2/JH Fusion Sequences in Follicular Lymphoma Patients : Comparison of Peripheral Blood and Bone Marrow Aspirate Samples

In patients with follicular lymphoma (FL), it is unresolved whether peripheral blood (PB) can replace bone marrow (BM) aspirate samples for detection of bcl-2/JH fusion sequences that result from the t(14;18)(q32;q21). We compare here the results of quantitative polymerase chain reaction (q-PCR) analysis for bcl-2/JH involving the major breakpoint cluster region (mbr) on paired PB and BM aspirate samples from 60 consecutive FL patients. There was a significant correlation between the level of bcl-2/JH fusion sequence obtained from PB and BM aspirate samples (r = 0.886), with 82% of samples showing less than one log of difference. Patients who had histological evidence of FL involving concurrent BM biopsy specimens had moderate to high levels of bcl-2/JH in both PB and BM aspirate samples, allowing unequivocal determination of translocation status (median bcl-2/JH to cyclophilin level was 8.014%). In contrast, patients with no detectable FL in their BM biopsy specimens often showed low levels of bcl-2/JH in both PB and BM aspirate samples (bcl-2/JH to cyclophilin median level = 0.006%), in a range similar to background levels that could be detected in patients without FL (n = 15, median bcl-2 mbr/JH to cyclophilin level = 0.002%). We conclude that PB can be used in place of BM aspirate samples to test for the presence of bcl-2 mbr/JH fusion sequence in FL patients and that either PB or BM aspirate testing yields a rough approximation of the degree of BM involvement by FL. However, in patients with minimal levels of bcl-2/JH in PB or BM aspirate samples, confirmation of this result by testing the primary tumor is recommended to confirm the presence of an identical bcl-2/JH fusion sequence and exclude false-positive results.

Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination.

We examined the influence of ROS on the phosphorylation and complex formation of Bcl-2 family proteins in Mn-superoxide dismutase (SOD) antisense-transfected squamous cell carcinoma cells, OSC-4 cells. The increase of intracellular ROS level induced by cis-diamminedichloroplatinum (CDDP) and gamma-ray treatment was greater in antisense-transfected cells than in control vector-transfected cells, and apoptosis was more extensively induced in the former. Antisense-transfected cells expressed high levels of Bax and Bak, but low levels of Bcl-2 and Bcl-XL when treated with CDDP, peplomycin, 5-fluorouracil or gamma-rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl-2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N-acetylcysteine and pyrrolidine dithiocarbamate)-pretreated control cells, but the phosphorylation levels were very low in the antisense-transfected cells. Bcl-2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense-transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl-2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression.