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[Cancer Research 59, 4119-4128, August 1, 1999]
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[Cancer Research 59, 4119-4128, August 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Expression Level of Bcl-2 Determines Anti- or Proapoptotic Function1

Nobusada Shinoura, Yoko Yoshida, Miyako Nishimura, Yukiko Muramatsu, Akio Asai, Takaaki Kirino and Hirofumi Hamada2

Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455 [N. S., Y. Y., M. N., Y. M., H. H.]; Department of Neurosurgery, Tokyo University, Tokyo 113 [N. S., A. A., T. K.]; and Core Research for Evolutional Science and Technology (CREST), Kawaguchi 332-0012 [T. K.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bcl-2 is an oncogene with antiapoptotic function. However, Bcl-2 is converted to a Bax-like death effector by caspases, suggesting that the expression of Bcl-2 may not favor the growth of cancers. We introduced the Bcl-2 gene to gliomas via adenovirus (Adv; Adv-Bcl-2) with the Adv for Fas (Adv-Fas) and the Adv for Fas ligand (Adv-FL) to evaluate the antiapoptotic function of Bcl-2. In U251 glioblastoma cells, Bcl-2 at a low level of expression repressed apoptosis induced by Adv-Fas and Adv-FL, whereas Bcl-2 at a high level of expression did not. On the other hand, Bcl-XL showed antiapoptotic function against Fas-mediated apoptosis, irrespective of its expression level. In glioblastoma cells, induction of Bcl-2 alone at a high level induced apoptosis, whereas induction of Bcl-XL alone did not. As the multiplicity of infection of Adv-Bcl-2 was increased, the quantity of a cleaved product of Bcl-2 increased. Induction of caspase-inhibitory genes (CrmA and p35) inhibited apoptosis induced by Adv-Bcl-2. Induction of Bcl-2 led to alteration of the membrane potential and structure of the mitochondria. In summary, although Bcl-2 at a low level of expression was antiapoptotic, Bcl-2 at a high level of expression was proapoptotic to Fas-mediated apoptosis. Overexpression of Bcl-XL was consistently antiapoptotic to Fas-mediated apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Bcl-2 gene, which is translocated to the heavy chain gene locus in B-cell follicular lymphoma and interferes with the cell death of lymphoma cells (1) , shows antiapoptotic function to various apoptosis-inducing stimuli such as growth factor deprivation (2 , 3) and anti-Fas antibody treatment (4) . However, clinical studies have shown that there is no correlation between the level of Bcl-2 expression and the aggressiveness of acute lymphoblastic leukemia (5) . Bcl-2 is expressed at a very low level in Kaposi’s sarcoma (6) . The expression of Bcl-2 suppresses the proliferation of developing B lymphocytes (7) and leukemia cells (8) by a cell cycle-inhibitory function, suggesting that overexpression of the Bcl-2 gene might delay the growth of cancer cells. Moreover, it has been shown that overexpression of the Bcl-2 protein increases the half-life of the Bax protein (9) and promotes the death of normal photoreceptor cells (10) , indicating that the Bcl-2 gene functions like a proapoptotic gene in some circumstances. It has recently been reported that caspase-3 cleaves Bcl-2 at Asp34 and transforms the Bcl-2 protein to an inducer of cell death (11) .

Some researchers (12 , 13) have reported that expression of Bcl-2 in gliomas is not related to malignant progression, whereas others have reported contradictory results (14) . In this study, we investigated apoptosis in glioma cells by overexpression of the Bcl-2 gene via a recombinant Adv3 vector. Here, we demonstrate that Bcl-2 has an antiapoptotic and a proapoptotic function, depending on the level of expression of Bcl-2, whereas Bcl-XL protects glioma cells from apoptosis irrespective of its level of expression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
The A-172 human glioma cell line was obtained from American Type Culture Collection (Manassas, VA). The U251 human glioma cell line was obtained from the Tumor Registry of the Division of Cancer Treatment, National Cancer Institute (Frederick, MD).

Adenoviral Vectors for Bcl-2, Bcl-XL, Fas, FL, CrmA, and p35.
The EcoRI fragment of human Bcl-2 cDNA from pB4 provided by Dr. Y. Tsujimoto (Osaka University Medical School, Suita, Japan; Ref. 1 ) was inserted into the EcoRI site of pCAcc (15) , which generated pCAcc-hBcl-2. The ClaI (blunt end) expression cassette from pCAcc-hBcl-2 was ligated to the SwaI site of cosmid pAxCALNLw (provided by Dr. I. Saito, University of Tokyo, Tokyo, Japan; Ref. 16 ), which resulted in the pAxCALNL-hBcl-2 cosmid. The on/off switching unit CALNL-hBcl-2 consisted of the CAG promoter, the neo gene, and polyadenylic acid sequence flanked by a pair of loxP sites, the Bcl-2 gene, and another polyadenylic acid signal (16) . CALNL-hBcl-2 without NCre expresses the neo gene but does not express the Bcl-2 gene. In the presence of NCre, which contains the Cre recombinase coding region (16) , the neo gene between the loxP sites is excised, and the CAG promoter and Bcl-2 gene are joined together, resulting in the Adv expressing the Bcl-2 gene under the control of the CAG promoter (16) .

The EcoRI fragment of human Bcl-XL cDNA from pSKIIhBcl-XL provided by Dr. L. H. Boise (University of Chicago, Chicago, IL; Ref. 17 ) was inserted into the EcoRI site of pCAcc, which generated pCAcc-hBcl-XL. The cosmid pAxCA-hBcl-XL was generated by inserting the ClaI expression cassette from pCAcc-hBcl-XL into the ClaI site of cosmid pAxcw (18) .

The HindIII/NsiI fragment of FLNL4 provided by Dr. E. Mita (Osaka University School of Medicine, Suita, Japan; Ref. 19 ) was inserted into the HindIII/PstI site of pBluescriptSKII+, generating pSKII+FL. The EcoRI (blunt end) fragment of human FL cDNA from pSKII+FL was inserted into the SwaI site of pAxCALNLw to generate pAxCALNL-hFL. To express the FL gene, the Cre/loxP system (16) was also used as described above for the Bcl-2 gene. Generation of the Adv for FL (Adv-FL) has been described previously (20) .

pRx-bsr plasmid DNA was constructed by introducing the blasticidin-selection marker gene, bsr (21) , at the 3' end of the internal ribosome entry site of the pRx retroviral vector (22) . The coding region of the cowpox virus caspase inhibitor CrmA gene, the 1176-bp fragment from the ATG start codon at NcoI to the 3' noncoding EcoRI, was obtained from pcDNA3-CrmA (23) . This was subcloned into the pRx-bsr plasmid, which generated pRx-CrmA-bsr. The XbaI/NotI (blunt end) fragment from pRx-CrmA-bsr was inserted into the SwaI site of pAxcw to generate pAxCA-CrmA.

The BamHI/EcoRI fragment (blunt end) of the baculovirus caspase inhibitor p35 gene from pcDNA-p35 provided by Dr. C. Kitanaka (National Cancer Research Institute, Tokyo, Japan; Ref. 24 ) was inserted into the SwaI site of pAxcw to generate pAxCA-p35.

Recombinant Advs were generated by cotransfection of the cosmids using the method described by Miyake et al. (18) . In particular, 293/CrmA cells, a subclone of 293 cells that had been transduced with the caspase-resistant CrmA gene, were used as host cells to generate the AxCA-hFas Adv (Adv-Fas), which has been described previously (25) . By performing a plaque assay using 293 cells, we determined the titers of the Adv, which were highly reproducible, suggesting that this method was reliable.

Adv-mediated gene transduction was performed using the method described by Yoshida et al. (15) . We used the Cre/loxP (16) system for Adv-Bcl-2 and Adv-FL. The Advs AxCALNL-hBcl-2 and AxCALNL-hFL (20) were always coinfected with the Adv for Cre recombinase (AxNCre; Ref. 16 , 20 ) at a MOI ratio of 2:1. The total MOI of Advs used to infect each cell was kept the same in all experiments by supplementing with the Adv for lacZ (Adv-lacZ; Ref. 15 ).

Assessment of Cell Death.
The degree of cell death was assessed by determining the percentage of cells that had died, the percentage of hypodiploid cells, and the degree of DNA fragmentation. The apoptotic cell death after infection with Adv-Fas started on day 2 and gradually increased on days 3 and 4. Therefore, we performed the quantitative assay for apoptosis on day 3.

To determine the percentage of cells that had died, cells that had adhered to the plate and those that had detached from the plate were stained with 0.2% trypan blue. The cells were then counted using a hemocytometer.

The percentage of hypodiploid cells was determined as described previously (20) . Briefly, ethanol-permeabilized cells were stained with PI and then analyzed with CELLQuest software using a FACScan (Becton Dickinson, San Jose, CA). The percentage of cells that had undergone apoptosis was assessed to be the ratio of the fluorescent area smaller than the G0-G1 peak to the total area of fluorescence. The average of the results from at least three samples of cells for each experimental condition is presented. The DNA fragments in apoptotic cells were also detected using the APO-BRDU kit (PharMingen, San Diego, CA) according to the manufacturer’s instructions. Briefly, the 3'-hydroxyl ends of DNA in apoptotic cells were labeled with Br-dUTPs by terminal deoxynucleotidyl transferase, and the Br-dUTP thus incorporated was identified by a FITC-labeled anti-bromodeoxyuridine monoclonal antibody. The samples were then stained with PI and analyzed by flow cytometry (FACS analysis).

For transmission electron microscopy, the cells were fixed in 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde at a pH of 7.5. They were then fixed in 0.1 M sodium phosphate buffer containing 1% OSO4 at a pH of 7.2. The cells were embedded into epon 812 (TAAB, Berkshire, United Kingdom). The ultrathin sections were contrasted with uranyl acetate and lead citrate and then examined with a H7000 transmission electron microscope (Hitachi Co., Tokyo, Japan).

Immunoblot Analysis.
Immunoblot analysis was performed using the enhanced chemiluminescence kit (Amersham, Buckinghamshire, England), as described previously (20) . Briefly, cells were washed with PBS and then scraped and centrifuged. After removal of the supernatant, the samples (2 x 106 cells) were resuspended in 100 µl of lysis buffer [10 mM Tris-HCl (pH 8.0), 0.2% NP40, and 1 mM EDTA], incubated on ice for 15 min, and centrifuged. The protein content of the supernatant was quantified by the DC protein assay kit (Bio-Rad, Hercules, CA), according to manufacturer’s instructions. An equal volume of 2x Laemmli buffer [0.125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% ß-mercaptoethanol, and 0.005% bromphenol blue] was added to the supernatant and boiled for 5 min. A total of 50–200 µg of protein were extracted from 2 x 106 cells. An equal amount of protein (10 µg) from each extract was separated on 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking the membrane with 5% dry milk in TBS [10 mM Tris-HCl (pH 7.5) and 150 mM sodium chloride], the membranes were incubated with each of the following primary antibodies overnight at 4°C: (a) mouse antihuman Bcl-2 monoclonal antibody (sc-509; Santa Cruz Biotechnology, Santa Cruz, CA); (b) mouse antihuman Bax monoclonal antibody (M010-3; Medical and Biological Laboratories, Nagoya, Japan); (c) goat antihuman caspase-3 polyclonal antibody (sc-1226; Santa Cruz Biotechnology); (d) rabbit antihuman PARP polyclonal antibody (SA252; Biomolecules for Research, Plymouth Meeting, PA); and (e) rabbit antihuman Bcl-X polyclonal antibody (B22630; Transduction Laboratories, Lexington, KY). After washing, the membranes incubated with the primary antibody for Bcl-2 or Bax were then incubated in 30 µl of horseradish peroxidase-conjugated rabbit antimouse IgG+A+M(H+L) (61-6420; Zymed, San Francisco, CA); the membranes incubated with the primary antibody for caspase-3 and Bcl-XL were then incubated in 30 µl of rabbit antigoat IgG(H+L) (Zymed), and the membranes incubated with the primary antibody for PARP were incubated in 30 µl of donkey antirabbit IgG(F(ab')2) (Amersham) using the enhanced chemiluminescence kit according to the manufacturer’s instructions (Amersham).

Mitochondrial Potential and Structure Analysis.
Variations in the mitochondrial transmembrane potential, {Delta}{Psi}m, during apoptosis induced by overexpression of Bcl-2 were studied using DiOC6 (D3652; Sigma, St. Louis, MO) as described previously (26) . U251 cells and A-172 cells were infected with either Adv-Bcl-2 or Adv-lacZ. Forty-eight h after infection, one million glioma cells were incubated in 1 ml of PBS containing 40 nM DiOC6 for 15 min at 37°C. DiOC6 membrane potential-related fluorescence was recorded using the FL1 photomultiplier setting in FACS analysis.

Alteration of the structure of the mitochondria during apoptosis was analyzed using NAO (A-1372; Molecular Probes, Eugene, OR), as described previously (26) . U251 cells and A-172 cells were infected with either Adv-Bcl-2 or Adv-lacZ. Forty-eight h after infection, one million glioma cells were incubated in 1 ml of PBS containing 100 nM NAO for 15 min at 37°C, followed by FACS analysis with the same setting as the {Delta}{Psi}m assessment.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antiapoptotic Function of Bcl-2 and Bcl-XL.
To evaluate the antiapoptotic function of Bcl-2 and Bcl-XL, we transduced Bcl-2 and Bcl-XL into U251 glioma cells at various expression levels via adenoviral vectors. U251 cells were coinfected with Adv-Fas (MOI, 10) together with either Adv-Bcl-2 or Adv-Bcl-XL (MOI, 0, 4, 20, and 100), and the resulting apoptosis was assayed for the percentage of dead cells (Fig. 1A)Citation and hypodiploid cells (Fig. 1B)Citation . The introduction of Fas via Adv induced apoptosis in gliomas, including U251 cells. At lower MOIs of 4 and 20, infection with Adv-Bcl-2 effectively repressed Adv-Fas-induced cytotoxicity (Fig. 1, A and B, a)Citation . It is noteworthy that the majority of cell death induced by Adv-Fas at a MOI of 10 was repressed by coinfection with Adv-Bcl-2 at a MOI of 4, at which the transduction efficiency evaluated by Adv-lacZ was only 10% (27) , possibly because 5-bromo-4-chloro-3-indolyl-ß-D-galactoside X-gal) staining for the evaluation of transduction efficiency in our experiment (15) was not sensitive enough to recognize all of the infected cells with one copy or a few copies of the lacZ reporter gene. Taken together, almost all of the U251 cells were transduced with the exogenous genes after infection with Adv at MOIs as low as 4 or 10.



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  		Fig. 1. A, the percentage of dead cells as measured by trypan blue exclusion 72 h after U251 cells were coinfected with Adv-Fas (MOI, 10) and Adv-Bcl-2 (MOI, 0, 4, 20, and 100; a) or with Adv-Fas (MOI, 10) and Adv-Bcl-XL (MOI, 0, 4, 20, and 100; b). Adv-lacZ was used to maintain a constant total MOI of Adv infected into the cells. The mean ± SD of the percentage of dead cells from the six preparations of each experimental condition is shown. B, FACS analysis of the percentage of hypodiploid cells 72 h after U251 cells were coinfected with Adv-Fas (MOI, 10) and Adv-Bcl-2 (MOI, 0, 4, 20, and 100; a) or with Adv-Fas (MOI, 10) and Adv-Bcl-XL (MOI, 0, 4, 20, and 100; b). Adv-Bcl-2 repressed Adv-Fas-induced apoptosis at MOIs of 4 and 20, but not at a MOI of 100. Adv-Bcl-XL repressed Adv-Fas induced apoptosis at MOIs of 4, 20, and 100. The mean ± SD of the percentage of hypodiploid cells in three preparations of each experimental condition is shown. C, flow cytometric analysis of Fas expression on the surface of U251 cells after infection with Adv-Fas alone or coinfection with Adv-Fas and Adv-Bcl-2. Flow cytometric analysis was performed 48 h after Adv infection. 1, U251 cells stained with isotype-matched control; 2, U251 cells stained with anti-Fas antibody; 3, U251 cells infected with Adv-Fas (MOI, 10) and stained with anti-Fas antibody; 4, U251 cells coinfected with Adv-Fas (MOI, 10) and Adv-Bcl-2 (MOI, 20) and stained with anti-Fas antibody. D, the percentage of dead cells (a) and hypodiploid cells (b) 72 h after U251 cells were coinfected with Adv-FL (MOI, 30) and Adv-Bcl-2 (MOI, 0, 4, and 100) or with Adv-FL (MOI, 30) and Adv-Bcl-XL (MOI, 0, 4, and 100). The mean ± SD of the percentage of dead cells from six preparations for each experimental condition is shown.

 
At a higher MOI of 100, Adv-Bcl-2 infection did not protect U251 cells from Adv-Fas-induced cytotoxicity (Fig. 1, A and B, a)Citation . The percentage of dead cells among U251 cells infected with Adv-Fas (MOI, 10) and Adv-Bcl-2 at low MOIs was relatively low (MOI of 4, 20 ± 2.8%; MOI of 20, 33 ± 5.4%), whereas the percentage of dead cells among U251 cells infected with Adv-Fas (MOI, 10) and Adv-Bcl-2 at a MOI of 100 was remarkably high (94 ± 3.3%; Fig. 1A, aCitation ). Similarly, the percentage of hypodiploid cells among U251 cells infected with Adv-Fas (MOI, 10) and Adv-Bcl-2 at low MOIs was relatively low (MOI of 4, 16 ± 0.9%; MOI of 20, 20 ± 2.5%), whereas the percentage of hypodiploid cells among U251 cells infected with Adv-Fas (MOI, 10) and Adv-Bcl-2 at a MOI of 100 was remarkably high (93 ± 0.2%). We evaluated the expression level of Fas after infection with Adv-Fas at a MOI of 10, with or without Adv-Bcl-2 at a MOI of 20. No difference in Fas expression levels was observed with or without Adv-Bcl-2 infection (Fig. 1C)Citation , indicating that the antiapoptotic effect of Adv-Bcl-2 at lower MOIs (4 and 20) was not due to the alteration of expression levels of surface Fas by coinfection with Adv-Bcl-2. Thus, a low-level expression of Bcl-2 was antiapoptotic, whereas a high level of Bcl-2 expression was proapoptotic to Fas-induced cytotoxicity in U251 cells. In contrast, Adv-Bcl-XL infection prevented Adv-Fas-mediated cell death of U251 cells in a MOI-dependent manner at all of the MOIs tested (Fig. 1, A and B, b)Citation . The assay for dead and hypodiploid cells was consistent with this observation (Fig. 1, A and B, b)Citation . The percentage of dead cells among U251 cells coinfected with Adv-Fas (MOI, 10) and Adv-Bcl-XL at MOIs of 4, 20, and 100 was 18 ± 2.7%, 13 ± 1.4%, and 10 ± 2.3%, respectively (Fig. 1A, b)Citation . The percentage of hypodiploid cells among U251 cells infected with Adv-Fas (MOI, 10) and Adv-Bcl-XL at MOIs of 4, 20, and 100 was 14 ± 0.8%, 1.9 ± 0.1%, and 2.2 ± 0.5%, respectively (Fig. 1B, b)Citation .

Introduction of FL via Adv induced apoptosis in gliomas, including U251 cells. U251 cells were also coinfected with Adv-FL (MOI, 30) together with either Adv-Bcl-2 or Adv-Bcl-XL (MOI, 0, 4, and 100). The resulting apoptosis was similar to that in the case of Adv-Fas. At a lower MOI of 4, infection with Adv-Bcl-2 effectively repressed Adv-FL-induced cytotoxicity (Fig. 1D)Citation . However, at a higher MOI of 100, Adv-Bcl-2 infection did not protect U251 cells from Adv-FL-induced cytotoxicity (Fig. 1D)Citation . The percentage of dead cells among U251 cells infected with Adv-FL (MOI, 30) and Adv-Bcl-2 at a MOI of 4 was relatively low (18 ± 2.3%), whereas the percentage of dead cells among U251 cells infected with Adv-FL (MOI, 30) and Adv-Bcl-2 at a MOI of 100 was remarkably high (94 ± 1.8%; Fig. 1D, aCitation ). Similarly, the percentage of hypodiploid cells among U251 cells infected with Adv-FL (MOI, 30) and Adv-Bcl-2 at a MOI of 4 was relatively low (7.4%), whereas the percentage of hypodiploid cells among U251 cells infected with Adv-Fas (MOI, 30) and Adv-Bcl-2 at a MOI of 100 was remarkably high (87%). Thus, a low-level expression of Bcl-2 was antiapoptotic, whereas a high level of Bcl-2 expression was proapoptotic to FL-induced cytotoxicity in U251 cells. In contrast, Adv-Bcl-XL infection prevented cell death of U251 cells induced by infection with adeno-FL, irrespective of the MOI of Adv-Bcl-XL infection (Fig. 1D)Citation . The percentage of dead cells among U251 cells infected with Adv-FL (MOI, 30) and Adv-Bcl-XL at MOIs of 4 and 100 was 15 ± 3.0% and 11 ± 3.1%, respectively (Fig. 1D, a)Citation . The percentage of hypodiploid cells among U251 cells infected with Adv-FL (MOI, 30) and Adv-Bcl-XL at MOIs of 4 and 100 was 8.3% and 4.1%, respectively (Fig. 1D, b)Citation .

Next we tested whether overexpression of Bcl-2 alone shows a proapoptotic effect. We used two glioma cell lines, U251 and A-172, that have different sensitivity to Fas-mediated apoptosis (20) . In A-172 cells, Adv infection did not induce toxicity at MOIs of up to 1000 (data not shown). U251 and A-172 cells were infected with various MOIs of either Adv-Bcl-2 or Adv-Bcl-XL. The MOI of Adv at which 50% and 100% of A-172 cells were infected with Adv-lacZ was 70 and 500, respectively; the MOI of Adv at which 50% and 100% of U251 cells were infected with Adv-lacZ was 20 and 100, respectively (27) . Thus, in this study, A-172 cells were infected with higher MOIs (MOIs of 100 and 500) than U251 cells (MOIs of 4, 20, and 100) to transduce cells with similar amounts of Bcl-2 and Bcl-XL proteins. In both the U251 and A-172 cell lines, as the MOI of Adv-Bcl-2 was increased, Adv-Bcl-2-induced cytotoxicity increased (Fig. 2A, b and e)Citation . In U251 cells infected with Adv-Bcl-2 at MOIs of 20 and 100, the percentage of hypodiploid cells was 39 ± 1.4% and 88 ± 0.9%, respectively (Fig. 2B, a)Citation . In A-172 cells infected with Adv-Bcl-2 at MOIs of 100 and 500, the percentage of hypodiploid cells was 18 ± 2.4% and 58 ± 2.1%, respectively (Fig. 2B, a)Citation . The titers of Adv carrying proapoptotic genes are sometimes underestimated when they prematurely kill the host 293 cells (25) . However, because we used the Cre/loxP system (16) for Adv-Bcl-2, which does not prematurely affect the viability of 293 cells, and because we consistently obtained considerably high titers of Adv-Bcl-2 viral solutions (4.5 x 109 pfu/ml), it is most probable that the titer of Adv-Bcl-2 is correctly estimated. In addition, the total MOI of Adv used to infect each cell was kept the same in all experiments by supplementing with the control Adv-lacZ to exclude the factor of infectivity (cytotoxicity) of Adv. Finally, infection with Adv did not show any cytotoxicity at up to a MOI of 400 in U251 cells or a MOI of 1000 in A-172 cells (20) . Taken together, it is concluded that these glioma cells underwent apoptosis not because of the cytotoxicity of Adv itself but because of Bcl-2 overexpression. On the other hand, Adv-Bcl-XL infection did not show a proapoptotic effect, even at high MOIs of Adv-Bcl-XL (Fig. 2A, c and f)Citation . The hypoploidy assay showed a low percentage of hypodiploid cells (<7%) in both cell lines, irrespective of the MOI of Adv-Bcl-XL (Fig. 2B, b)Citation .



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  		Fig. 2. A, phase-contrast microscopic photographs of U251 cells and A-172 cells examined 72 h after infection with Adv (original magnification, x100). Adv-lacZ was used to maintain a constant total MOI of Adv infected into the cells. a, U251 cells infected with Adv-lacZ (MOI, 150); b, U251 cells infected with Adv-Bcl-2 (MOI, 100) and Adv-NCre (MOI, 50); c, U251 cells infected with Adv-Bcl-XL (MOI, 100) and Adv-lacZ (MOI, 50); d, A-172 cells infected with Adv-lacZ (MOI, 750); e, A-172 cells infected with Adv-Bcl-2 (MOI, 500) and Adv-NCre (MOI, 250); f, A-172 cells infected with Adv-Bcl-XL (MOI, 500) and Adv-lacZ (MOI, 250). Infection with Adv-Bcl-2 induced apoptosis in U251 and A-172 cells, whereas infection with Adv-Bcl-XL did not induce apoptosis. B, FACS analysis of the percentage of hypodiploid cells 72 h after U251 cells and A-172 cells were infected with Adv-Bcl-2 (a) or Adv-Bcl-XL (b). Infection with Adv-Bcl-2 alone induced apoptosis in U251 cells (at MOIs of 20 and 100) and in A-172 cells (at MOIs of 100 and 500). Infection with Adv-Bcl-XL alone did not induce apoptosis in U251 cells or A-172 cells. The mean ± SD of the percentage of hypodiploid cells in three preparations for each experimental condition is shown.

 
Apoptosis induced by overexpression of Bcl-2 was also demonstrated by the assay for incorporation of Br-dUTP (Fig. 3A)Citation and transmission electron microscopy (Fig. 3B)Citation . Apoptotic cells were detected by labeling the 3'-hydroxyl ends of DNA with Br-dUTP; they were then allowed to bind with FITC-labeled anti-bromodeoxyuridine monoclonal antibody and analyzed by FACS analysis. Fragmented DNA appear in the upper left and upper right quadrants of the FACS analysis (Fig. 3A)Citation . Seventy-two h after infection, a higher percentage of fragmented DNA was present in the U251 cells infected with Adv-Bcl-2 (MOI, 100; 89%) than in the U251 cells infected with Adv-lacZ (0.1%; Fig. 3ACitation ). A small population of A-172 cells infected with control Adv-lacZ (2.4%; upper left and upper right quadrants) showed a mild uptake of Br-dUTP, possibly because of the small but significant damage induced by a high MOI (750) of Adv infection (Fig. 3A)Citation . However, the percentage of A-172 cells in the upper left quadrant, which represents the hypodiploid cells with fragmented DNA, increased from 0.3% to 6.3% by infection with Adv-Bcl-2 compared with Adv-lacZ (Fig. 3A)Citation . This indicates that infection with Adv-Bcl-2 induced significant apoptosis in A-172 cells, although A-172 cells were less sensitive to apoptosis induced by Bcl-2 overexpression. This is in accord with our previous findings that A-172 cells were relatively more resistant to apoptosis induced by proapoptotic genes such as FL than U251 cells (20) .



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  		Fig. 3. A, FACS analysis of the percentage of apoptotic cells 72 h after U251 cells and A-172 cells were infected with Adv-Bcl-2 (MOI of 100 for U251 cells; MOI of 500 for A-172 cells) or Adv-lacZ, as described in "Materials and Methods." The X axis represents PI-related fluorescence, and the Y axis represents Br-dUTP-related fluorescence. The points in the upper left and right quadrants represent apoptotic cells. Infection of U251 and A-172 cells with Adv-Bcl-2 induced apoptosis, whereas infection of the cells with Adv-lacZ did not induce apoptosis. B, ultrastructural analysis of U251 cells and A-172 cells examined 96 h after infection with Adv-Bcl-2. a and b, U251 cells infected with Adv-Bcl-2 (MOI, 100; a, x6000; b, x5000); c and d, A-172 cells infected with Adv-Bcl-2 (MOI, 500; c, x5000; d, x12000). Apoptotic bodies (a and c), condensed chromatin (b), and mitochondrial hydropsy (d) can be seen.

 
To examine the morphology of the Bcl-2-induced cellular damage, we performed electron microscopic analyses. Because the degree of the typical apoptotic cellular damage became much pronounced on day 4 after Adv-Bcl-2 infection, we demonstrate the electron microscopic features of the cells on day 4 in Fig. 3BCitation . U251 cells and A-172 cells examined by transmission electron microscopy 96 h after infection with Adv-Bcl-2 exhibited the typical ultrastructural features of apoptosis (Fig. 3B)Citation . Many cells were in various stages of the apoptotic process and were characterized by condensation of the heterochromatin (Fig. 3B, b)Citation , fragmentation of the heterochromatin and cytoplasm to form apoptotic bodies (Fig. 3B, a and c)Citation , and mitochondrial hydropsy (Fig. 3B, d)Citation .

Immunoblot Analysis of Bcl-2, Bax, Caspase-3, PARP, and Bcl-XL.
After the induction of apoptosis, Bcl-2 is cleaved by caspase-3 in cells that overexpress caspase-3, and the Bcl-2 cleavage product further activates the caspases (11) . Thus, we investigated whether Bcl-2 itself is cleaved by overexpression of Bcl-2 with simultaneous activation of caspase-3 and cleavage of PARP. Expression of Bcl-2, caspase-3, and PARP was analyzed in A-172 and U251 cells. Fig. 4ACitation shows the dose response of Bcl-2 in U251 and A-172 cells. The apparent molecular weight of Bcl-2 was 26,000 (Fig. 4A)Citation . In addition to the Mr 26,000 Bcl-2 protein (p26), a Mr 20,000 band (p20) was observed in the U251 cells infected with Adv-Bcl-2 and in the A-172 cells infected with Adv-Bcl-2; both of these cell lines become apoptotic with the overexpression of Bcl-2 (Fig. 4ACitation , Lane 4 in a and Lanes 3 and 4 in b). It is noteworthy that the intensity of the bands p26 and p20, which was detected by anti-Bcl-2 monoclonal antibody and hence represented the expression of Bcl-2, increased in a MOI-dependent manner (Fig. 4A)Citation . The p20 band of Bcl-2 (Fig. 4A)Citation very likely corresponds to the previously reported p23, which is the cleaved form of Bcl-2 and has a function similar to that of Bax (11) . In U251 cells and A-172 cells infected with Adv-Bcl-2 at two different MOIs (U251 cells, MOI of 20 and 100; A-172 cells, MOI of 100 and 500), the quantity of p20 increased at higher MOIs of Adv-Bcl-2, whereas the quantity of p26 remained at a similar level (Fig. 4A, a and b)Citation . The endogenous expression levels of p26 in several glioma cell lines including U251 and A-172 were lower compared with those after infection with Adv-Bcl-2 at a MOI of 20 (Fig. 4A)Citation . The p20 Bcl-2 cleavage product may have a proapoptotic function and may accelerate cell death. We assessed the level of Bax expression in U251 and A-172 cells that were and were not infected with Adv-Bcl-2. The level of Bax expression 48 h after U251 cells were infected with Adv-Bcl-2 did not differ from that in U251 cells not infected with Adv-Bcl-2 (Fig. 4B, a)Citation . This suggests that the apoptosis induced by overexpression of Bcl-2 is not induced through the induction of the Bax gene. The active form of caspase-3, p17, was present in U251 cells infected with Adv-Bcl-2 (Fig. 4B, bCitation , Lanes 3 and 4), although p17 was not present in U251 cells that were not infected with Adv-Bcl-2 (Fig. 4B, bCitation , Lane 1) or in U251 cells infected with Adv-lacZ (Fig. 4B, bCitation , Lane 2). In our analyses of cellular proteins, we extracted proteins from U251 cells on day 2 after infection with Adv-Bcl-2 because apoptotic cellular damage on day 3 was so severe that it hampered the analyses of the earlier apoptotic cellular changes. On day 3 after Adv-Bcl-2 infection (MOI, 20), caspase-3 should be activated because apoptosis was inhibited by CrmA and p35 on the same day (Fig. 5)Citation . It suggests that in U251 cells, the cleaved product of Bcl-2, p20 (Fig. 4A, a)Citation , or the cleaved product of caspase-3, p17 (Fig. 4B, b)Citation , which appeared faintly on day 2, would appear more clearly on day 3, resulting in the increased degree of apoptosis. These results indicate that overexpression of Bcl-2 activates caspase-3, which in turn cleaves and thereby activates apoptosis executor molecules. The cleaved product of PARP (p85; Fig. 4B, cCitation ) was also present in higher amounts in U251 cells infected with Adv-Bcl-2 at a MOI of 100 (Fig. 4B, cCitation , Lane 4) than in U251 cells infected with Adv-Bcl-2 at a MOI of 20 (Fig. 4B, cCitation , Lane 3).



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  		Fig. 4. A, immunoblot analysis of Bcl-2 extracted from U251 cells and from A-172 cells 48 h after infection with Adv. Adv-lacZ was used to maintain a constant total MOI of Adv infected into the cells. a, Lane 1, U251 cells; Lane 2, U251 cells infected with Adv-lacZ (MOI, 150); Lane 3, U251 cells infected with Adv-Bcl-2 (MOI, 20), Adv-NCre (MOI, 10), and Adv-lacZ (MOI, 120); Lane 4, U251 cells infected with Adv-Bcl-2 (MOI, 100) and Adv-NCre (MOI, 50). b, Lane 1, A-172 cells; Lane 2, A-172 cells infected with Adv-lacZ (MOI, 750); Lane 3, A-172 cells infected with Adv-Bcl-2 (MOI, 100), Adv-NCre (MOI, 50), and Adv-lacZ (MOI, 600); Lane 4, A-172 cells infected with Adv-Bcl-2 (MOI, 500) and Adv-NCre (MOI, 250). In both the A-172 cells and U251 cells, infection with Adv-Bcl-2 induced the expression of p20 in a MOI-dependent manner. B, immunoblot analysis of (a) Bax, (b) caspase-3, and (c) PARP extracted from U251 cells 48 h after infection with Adv. Adv-lacZ was used to maintain a constant total MOI of Adv infected into the cells. a—c, Lane 1, U251 cells; Lane 2, U251 cells infected with Adv-lacZ (MOI, 150); Lane 3, U251 cells infected with Adv-Bcl-2 (MOI, 20), Adv-NCre (MOI, 10), and Adv-lacZ (MOI, 120); Lane 4, U251 cells infected with Adv-Bcl-2 (MOI, 100) and Adv-NCre (MOI, 50). Bax expression was not affected by infection with Adv-Bcl-2. The active form of caspase-3 (p17) and the cleaved products of PARP (p85) were present in the cells infected with Adv-Bcl-2 in a MOI-dependent manner. C, immunoblot analysis of Bcl-XL extracted from U251 cells 48 h after infection with Adv. Adv-lacZ was used to maintain a constant total MOI of Adv infected into the cells. Lane 1, U251 cells; Lane 2, U251 cells infected with Adv-lacZ (MOI, 20); Lane 3, U251 cells infected with Adv-Bcl-XL (MOI, 20).

 


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  		Fig. 5. FACS analysis of the percentage of hypodiploid cells 72 h after U251 cells were coinfected with (A) Adv-Bcl-2 (MOI, 20) and Adv-CrmA (MOI, 0, 100, and 300) or (B) Adv-Bcl-2 (MOI, 20) and Adv-p35 (MOI, 0, 100, and 300). Adv-Bcl-2-induced apoptosis was repressed by coinfection with Adv-CrmA and by coinfection with Adv-p35. The mean ± SD of the percentage of hypodiploid cells in three preparations for each experimental condition is shown.

 
In contrast with the case for Bcl-2, after infection with Adv-Bcl-XL at a MOI of 20, the exogenous expression level of Bcl-XL was highly increased compared with the endogenous expression level of Bcl-XL in U251 cells, without any evidence of cleaved product (Fig. 4C)Citation .

Inhibition of Apoptosis by CrmA and p35.
CrmA and p35 inhibit the protease activities of caspases (28, 29, 30, 31) . We further assessed the involvement of caspases in the apoptosis induced by Adv-Bcl-2 using these caspase inhibitors. Either the Adv for CrmA (Adv-CrmA) or the Adv for baculovirus p35 (Adv-p35) was coinfected with Adv-Bcl-2, and the resulting apoptosis was assayed by detecting hypodiploid cells using a FACS analysis. Adv-CrmA infection effectively repressed apoptosis induced by Adv-Bcl-2 (MOI, 20). The percentage of hypodiploid cells among U251 cells infected with Adv-Bcl-2 alone was 34 ± 1.1% (Adv-CrmA, MOI of 0). In U251 cells coinfected with Adv-Bcl-2 (MOI, 20) and Adv-CrmA at MOIs of 100 and 300, the percentage of hypodiploid cells was 7.3 ± 0.6% and 11 ± 0.6%, respectively (Fig. 5A)Citation . Similarly, infection with Adv-p35 repressed apoptosis induced by Adv-Bcl-2 (MOI, 20). The percentage of hypodiploid cells among U251 cells infected with Adv-Bcl-2 alone was 34 ± 1.1% (Adv-p35, MOI of 0), whereas the percentage of U251 cells coinfected with Adv-Bcl-2 (MOI, 20) and Adv-p35 at MOIs of 100 and 300 was 24 ± 0.6% and 11 ± 1.9%, respectively (Fig. 5B)Citation . These results indicate that caspases, especially caspase-1 and/or caspase-8 (28 , 31) , are involved in the Bcl-2-induced apoptotic pathways.

Alteration of Mitochondrial Structure and Function by Overexpression of Bcl-2.
To investigate the modification of mitochondrial function as a result of Bcl-2-induced apoptosis, we analyzed the uptake of cationic lipophilic dye DiOC6, which is associated with mitochondrial membrane potential ({Delta}{Psi}m; Ref. 32 ), by FACS analysis. Two days after infection with Adv-Bcl-2, the {Delta}{Psi}m-related fluorescence, DiOC6, of U251 cells infected with Adv-Bcl-2 and A-172 cells infected with Adv-Bcl-2 was less than that of the respective cells infected with Adv-lacZ (Fig. 6A)Citation . We also evaluated the structure of the mitochondria using the NAO incorporation assay. Two days after infection with Adv, U251 and A-172 cells were treated with NAO, and the mitochondrial structure of U251 and A-172 cells infected with Adv-Bcl-2 and those infected with Adv-lacZ was assessed by FACS analysis. If the NAO-related fluorescence of the U251 cells and A-172 cells infected with Adv-Bcl-2 was less than that of the respective cells infected with Adv-lacZ, the cells infected with Adv-Bcl-2 have a decreased amount or drastic alteration of the cardiolipin molecule, a molecule restricted to the inner mitochondrial membrane (33) . The NAO-related fluorescence of U251 and A-172 cells infected with Adv-Bcl-2 was much less than that of the respective cells infected with Adv-lacZ (Fig. 6B)Citation . These results indicate that apoptosis by overexpression of Bcl-2 is associated with the alteration of the membrane potential and structure of the mitochondria, i.e., mitochondrial damage. The data from the NAO incorporation assay are in accord with the ultrastructural observation of severe mitochondrial damage in the apoptotic U251 and A-172 cells that overexpress Bcl-2 (Fig. 3B, d)Citation .



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  		Fig. 6. A, FACS analysis of the mitochondrial membrane potential ({Delta}{Psi}m) of apoptotic cells 48 h after U251 cells and A-172 cells were infected with Adv-Bcl-2 (MOI of 100 for U251 cells; MOI of 500 for A-172 cells) or Adv-lacZ as described in "Materials and Methods." Note that the population with low {Delta}{Psi}m increased in the glioma cells through infection with Adv-Bcl-2, compared with the respective cells through infection with control Adv-lacZ. This indicates that the {Delta}{Psi}m had decreased drastically in the cells infected with Adv-Bcl-2. B, FACS analysis of the structure of the mitochondrial membrane of apoptotic cells 48 h after U251 cells and A-172 cells were infected with Adv-Bcl-2 (MOI of 100 for U251 cells; MOI of 500 for A-172 cells) or Adv-lacZ as described in "Materials and Methods." Note that the population with low NAO-related fluorescence increased in the glioma cells through infection with Adv-Bcl-2, as compared with the respective cells through infection with control Adv-lacZ. This indicates that the structure of the mitochondrial membrane is less intact in the U251 cells and A-172 cells infected with Adv-Bcl-2.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that Bcl-2 protected U251 cells against Fas-mediated apoptosis less effectively compared with Bcl-XL, and excessive expression of Bcl-2 in gliomas induces apoptosis. Bcl-2 at a high expression level induced apoptosis. Apoptosis was associated with an increase in p20, which is the cleaved form of Bcl-2 and possibly corresponds to a Bax-like cytotoxic effector (11) . Similar results were obtained in U251 cells treated with other proapoptotic stimuli, e.g., FL overexpression (Fig. 1D)Citation , and in nerve growth factor-treated PC12 cells transduced with other proapoptotic genes, e.g., Bax.4 In nerve growth factor-treated PC12 cells, Bcl-XL showed more efficient antiapoptotic function than Bcl-2, and overexpression of Bcl-2 induced apoptosis. In addition, immunoblot analysis revealed that the cleaved product of Bcl-2, p20, appeared after infection with Adv-Bcl-2 at a higher MOI. One or several caspases (caspase-1, -3, or -8) are involved in apoptosis induced by Bcl-2 overexpression. The function and structure of the mitochondria change during the progression of apoptosis. Bcl-2 is reported to inhibit the release of apoptogenic proteases (apoptosis-inducing factor) and cytochrome c from the mitochondria (34, 35, 36) . In this study, Bcl-2 at a low expression level did indeed inhibit the proapoptotic function of Fas. However, overexpression of Bcl-2 quickly and drastically induced apoptosis in U251 cells. This suggests that Bcl-2 overexpression readily activated the proapoptotic cascades, resulting in the quick cleavage of Bcl-2. Overexpression of Bcl-2 would cause mitochondrial damage, namely, mitochondrial permeability transition pore (37) , followed by the activation of caspases, possibly by the release of cytochrome c (35 , 36) and apoptogenic proteases (34) from the mitochondria. The activated caspases cleaved Bcl-2 (11) into a Bax-like cytotoxic effector, which in turn accelerated cytotoxicity (38) , possibly by further damaging the mitochondrial membrane.

By what mechanism does overexpression of Bcl-2 damage mitochondrial function and structure? Schendel et al. (39) suggested that Bcl-2 forms ion channels that allow the transport of an ion or a protein across the mitochondrial membrane in the direction that is presumably cytoprotective. Alternatively, it was suggested that Bcl-2 forms cytotoxic channels, and heterodimerization of Bcl-2 and Bax protects cells by nullifying the channel activity (40) . In Ref. 40 , it is suggested that both Bcl-2 and Bax might form cytotoxic channels in cells, with Bcl-2/Bax heterodimerization nullifying channel activity and thus promoting cell survival. To test this hypothesis, we performed an assay for apoptosis in U251 cells after infection with Adv-Bax and/or Adv-Bcl-2, and we found that Bax expression does not suppress Bcl-2-induced apoptosis (data not shown). Thus, our preliminary result suggests that heterodimerization of Bcl-2 and Bax does not prevent Bcl-2-induced apoptosis. However, when Bcl-2 expression is not high enough to induce apoptosis, Bcl-2 repressed Bax-induced apoptosis by heterodimerization of Bcl-2 and Bax (41) . Additional studies are required to elucidate the molecular mechanisms of the anti- and proapoptotic functions of Bcl-2.

In clear contrast to Bcl-2, overexpression of Bcl-XL did not induce cellular damage. Indeed, Bcl-XL is distributed in the mitochondrial membrane, and has an antiapoptotic function. The level of Bcl-XL expression is quite different in many tissues and cell lines. Bcl-XL is predominantly expressed in a number of cancer cells, such as Kaposi’s sarcoma (6) , gastric cancers (42) , and leukemia (43) . An apoptosis-resistant variant of leukemia HL-60 cells switched expression from the antiapoptotic protein Bcl-2 to Bcl-XL (44) . Bcl-XL is expressed at higher levels than Bcl-2 in proliferating cells such as regenerating liver cells (45) and primitive hematopoietic stem cells (46) . In comparison with the antiapoptotic function of Bcl-2, Bcl-XL more markedly protects lymphoid cells from chemotherapy (47) , B cells from immunosuppression-induced apoptosis (48) , COS-7 cells from Bax-induced apoptosis (23) , and glioma cells from Fas-mediated apoptosis in this report. Bcl-2 and Bcl-XL may have distinct functional roles in the survival and/or growth of individual cancers as well as normal tissues.

What is the functional role of Bcl-2 in cells? In this study, Bcl-2 was demonstrated to be antiapoptotic at lower expression levels. The protein level of Bcl-2 introduced by infection with Adv-Bcl-2 at higher MOIs of 20 or 100 might be higher than the physiological level of expression of Bcl-2. Indeed, all of the glioma cell lines such as A-172, U-87MG, U251, and U-373MG show a lower expression of Bcl-2 compared with that after infection with Adv-Bcl-2 at a MOI of 20 or 100. The levels of Bcl-2 transduced after infection with Adv-Bcl-2 might not be similar to the endogenous cellular Bcl-2 levels, implying that the cells are not able to express Bcl-2 protein over the levels obtained after infection with Adv-Bcl-2 at a MOI of 20 or 100 due to apoptosis. In other words, if the cells express Bcl-2 protein at a level greater than the physiological level, they would undergo apoptosis and therefore are unable to express the Bcl-2 protein at higher levels. Among nonlymphoid tissues, Bcl-2 is expressed in differentiating epithelia such as the skin and intestines and in long-lived postmitotic cells such as neurons (49) . Follicular lymphoma and chronic lymphocytic leukemia, which are associated with chromosomal translocation of the Bcl-2 gene and unregulated expression of Bcl-2, predominantly contain quiescent, noncycling tumor cells (50) . The Bcl-2 protein has a cell cycle-inhibitory function and reduces proliferation (7 , 8) . Thus, an appropriate but relatively low expression level of Bcl-2 may be associated with longevity in normal tissues.

Several therapeutic approaches using the antiapoptotic function of Bcl-2 have been reported. Treatment with overexpression of Bcl-2 has been tried to save neurons from ischemia (51, 52, 53) , neurotoxin (54) , injury (55) , and amyotrophic lateral sclerosis (56) . Bcl-2 was also useful for saving pancreatic ß cells from destruction in autoimmune diabetes (57) . In this study, we demonstrated that Bcl-2 and Bcl-XL have different antiapoptotic functions in cancers, depending on the level of expression. However, it remains possible that Bcl-2 and Bcl-XL have different functions in normal differentiated cells and in cancer cells. Indeed, in our preliminary experiments, overexpression of Bcl-2 induced apoptosis at lower levels in differentiated PC-12 cells compared with that in glioma cells.4 Further investigation is needed to test the antiapoptotic effect of Bcl-2 and Bcl-XL in various cancers and normal tissues. Our results indicate that we should carefully determine the appropriate antiapoptotic gene and its expression level to develop therapeutic approaches for the protection of individual tissues by evaluating the antiapoptotic effects of Bcl-2 as well as Bcl-XL.


   ACKNOWLEDGMENTS
 
We thank Dr. S. Fukuda for technical assistance with the electron microscopic study and Dr. H. Shinoura for assistance in the preparation of this manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Culture and Science of Japan and by grants from the Ministry of Health and Welfare of Japan. Back

2 To whom requests for reprints should be addressed, at Cancer Chemotherapy Center, Cancer Institute, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Phone: 81-3-3918-0111; Fax: 81-3-3918-3716; E-mail: hhamada{at}jfcr.or.jp Back

3 The abbreviations used are: Adv, adenovirus; FL, Fas ligand; MOI, multiplicity of infection; Br-dUTP, bromolated dUTP nucleotide; PI, propidium iodide; PARP, poly(ADP ribose) polymerase; DiOC6, 3,3'-dehexyloxacarbocyanine iodide; NAO, nonyl acridine orange; FACS, fluorescence-activated cell-sorting. Back

4 Unpublished observations. Back

Received 10/27/98. Accepted 6/17/99.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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