Resistance of pleural mesothelioma cell lines to apoptosis: relation to expression of Bcl-2 and Bax

Sudha Rani Narasimhan1, Lin Yang1, Brenda I. Gerwin2, and V. Courtney Broaddus1

1 Department of Medicine and Lung Biology Center, San Francisco General Hospital, San Francisco, California 94110; and 2 Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

A failure of normal apoptosis, often due to mutant p53, may contribute to the formation of a cancer and to its resistance to therapy. Mesothelioma, an asbestos-induced tumor, is highly resistant to therapy but generally expresses wild-type p53. We asked whether mesothelioma was resistant to apoptosis and whether resistance was associated with altered expression of the antiapoptotic protein Bcl-2 or proapoptotic protein Bax. We found that three mesothelioma cell lines (1 with wild-type p53) were highly resistant to apoptosis induced by oxidant stimuli (asbestos, H2O2) or nonoxidant stimuli (calcium ionophore) compared with primary cultured mesothelial cells. By immunostaining, one of these three lines expressed Bcl-2 but only during mitosis. By immunoblotting, 3 of 14 additional mesothelioma lines (9 of 14 with wild type p53) expressed Bcl-2 but all 14 of 14 expressed the proapoptotic Bax, giving a low ratio of Bcl-2 to Bax. We conclude that mesothelioma cell lines are resistant to apoptosis and that the failure in apoptosis is not explained by Bcl-2 but by other mechanisms that counteract the proapoptotic effect of Bax.

mesothelial cell; p53; reactive oxygen species; asbestos; cancer

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

RESISTANCE TO APOPTOSIS may be important both for the initial development and for the continuing survival of tumors. An initial resistance to apoptosis may be necessary to allow the amplification of a population of abnormal cells (42), and a continued resistance to apoptosis may underlie the insensitivity of tumor cells to chemotherapy and radiotherapy (43). Mesothelioma, a tumor notoriously unresponsive to chemotherapy and radiotherapy (4, 34), has not been studied for its resistance to apoptosis. Resistance to apoptosis could explain the overall insensitivity of mesothelioma to therapy; understanding the means of tumor resistance to apoptosis could lead to novel treatments or to ways of increasing the sensitivity to standard treatments.

Mesothelioma is induced by asbestos fibers, presumably in their direct interaction with mesothelial cells (30). When asbestos fibers are added to cultured mesothelial cells, we and others have shown that a large percentage of those mesothelial cells undergo apoptosis (2, 8), in large part due to reactive oxygen species and DNA damage (8). Interference with an apoptotic response to asbestos could lead to an accumulation of cells with asbestos-induced DNA damage and, ultimately, through a multistep process of accumulated mutations, to mesothelioma (8). Therefore, it is possible that mesothelioma arises in part because of an initial selective resistance to asbestos-induced apoptosis. Indeed, in earlier studies investigating cytotoxicity by measuring colony-forming efficiency and clonal growth rates, mesothelioma cell lines had shown a selective resistance to asbestos (15). Mesothelioma cell lines have not been tested for their resistance to apoptosis, either generally to diverse stimuli or specifically to asbestos.

Because mesothelioma has mostly been found to have wild-type p53 (27, 29), a major regulator of apoptosis, its resistance to apoptosis would be expected to arise downstream from p53. In some tumors with wild-type p53, the downstream abnormality has been located in the Bcl-2 family of proteins, a family of proteins regulating cell death (35). Overexpression of Bcl-2, a protein that suppresses apoptosis from a wide variety of stimuli, including reactive oxygen species, chemotherapeutic drugs, and irradiation (14, 36), has been found to correlate with poor prognosis in several tumors such as breast, ovarian, prostate, and lung cancer (19, 21, 25, 40). Bcl-2 functions by heterodimerizing with Bax, a protein that accelerates apoptosis (33, 46). Thus a ratio of Bcl-2 to Bax greater than one may correlate with resistance to apoptosis. Indeed, a high ratio of Bcl-2 to Bax correlates with poor prognosis and poor histological grade in tumors such as neuroendocrine lung cancers (6) and bladder cancer (17), whereas a low ratio of Bcl-2 to Bax is found in cell lines derived from highly responsive tumors such as testicular cancer (11). In histological reports of mesothelioma, Bcl-2 has not been found to be overexpressed, although Bax expression was not examined (9, 39). Because Bcl-2 overexpression or Bax underexpression could account for a resistance to apoptosis even in the presence of a wild-type p53, we considered this family of proteins as possible oncogenic candidates in mesothelioma.

In this study, we asked whether mesothelioma is resistant to apoptosis, and if so, was the resistance general to different types of apoptosis or specific to asbestos-induced apoptosis. To test cellular responses to different apoptotic stimuli, we used three cell lines derived from mesothelioma (1 with wild-type p53 and 2 with an abnormal p53) and compared their responses with those of primary cultured mesothelial cells. We also used mesothelial cell lines developed with or without exposure to asbestos to learn whether they exhibited a selective resistance to asbestos-induced apoptosis. Finally, we analyzed the three mesothelioma-derived cell lines for Bcl-2 expression by immunocytochemistry and the lysates of these and 11 additional mesothelioma cell lines, most of which express wild-type p53 (27), for expression of Bcl-2 and Bax.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cells. The mesothelioma-derived cell lines were obtained as follows: HuT 28 with wild-type p53 from Dr. Brenda Gerwin (National Cancer Institute, Bethesda, MD; see Ref. 27) and LRK1A and REN with abnormal p53 resulting in an absence of p53 protein from Dr. Steven Albelda (Univ. of Pennsylvania, Philadelphia, PA and personal communication). Primary cultured rabbit pleural mesothelial cells (passages 5-7) were obtained from healthy rabbits, and primary cultured human cells (passages 2-5) were obtained from pleural and peritoneal liquids from patients with nonmalignant diseases, as described previously (5). An SV40 transformed human mesothelial line, MeT-5A, was obtained from American Type Culture Collection. Three human mesothelial cell lines were previously shown to have a different exposure history and resistance to asbestos: the asbestos-exposed and resistant line P19A/T, which was established by transfecting an asbestos-exposed cell line, P19, with the SV40 T cell antigen, and the more sensitive lines MEJ-T10 and MeT-34, which are tumorigenic lines derived from MeT-5A, the first transfected with EJ-ras p21 and the second transfected with platelet-derived growth factor-A (15). Lysates were obtained from the following 11 additional mesothelioma cell lines: VAMT-1, M24K, M20, M14K, M9K, M28, DND, M33K, and M25K with wild-type p53 and M15 and JMN with p53 mutations (27).

Tumor cell lines were cultured in RPMI 1640 medium supplemented with HEPES (10 mM), 10% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT), L-glutamine (2 mM; GIBCO, Grand Island, NY), penicillin (100 U/ml; GIBCO), and streptomycin (100 µg/ml; GIBCO). The primary cultured rabbit cells were grown in RPMI 1640-DMEM supplemented with the above components. The primary cultured human cells were grown in LHC-MM medium (Biofluids, Rockville, MD), L-Glutamax II (2 mM; GIBCO), penicillin, streptomycin, and Fungizone (2.5 µg/ml; GIBCO).

Reagents. Crocidolite asbestos fibers, an amphibole fiber, were obtained from the National Institute of Environmental Health and Safety (Research Triangle Park, NC). Acridine orange and calcium ionophore A-23187 were from Sigma Chemical (St. Louis, MO). The H2O2 was obtained from Fisher Scientific (Pittsburgh, PA).

Detection of apoptosis. Apoptosis was quantified by two methods: the detection of acridine orange-stained condensed nuclei by fluorescent microscopy and the detection of surface expression of phosphatidylserine by flow cytometry, as we have done previously (8). For both assays, subconfluent cells (70-80% confluent) in six-well uncoated plates were exposed to apoptotic stimuli for 18 h (10 and 20 µg/cm2 crocidolite, 30 and 100 µM H2O2, and 0.5 µM calcium ionophore). Cells were then detached with trypsin (0.25%) and EDTA (0.5 mM) and combined with floating cells. For acridine orange staining, cells (5 × 105) were stained with acridine orange (10 µg/ml) for 4 min, fixed with glutaraldehyde (2.5%, vol/vol; Sigma) for 30 min in the dark, and then dropped onto glass slides, covered with coverslips, and sealed. Apoptotic cells were counted as a percentage of total cells in a blinded fashion with epifluorescence microscopy.

For detection of phosphatidylserine on the outer leaflet of cell membranes as evidence of a loss of membrane asymmetry characteristic of apoptosis, cells were stained with a green fluorescent protein (GFP)-annexin V fusion protein and analyzed by flow cytometry (22). Cells (5 × 105) were resuspended in HEPES buffer with 2 mM CaCl2, incubated with GFP-annexin V fusion protein (3 µg/ml in HEPES) for 10 min on ice, washed, and analyzed by flow cytometry using a FACSort flow cytometer (Becton Dickinson, San Jose, CA). The GFP-annexin V fusion protein was constructed and purified as described (13). GFP-annexin V had identical properties as the FITC-annexin V we have used previously (8). Binding specificity was confirmed by a lack of binding in calcium-free buffers and by competition with unlabeled annexin V. Because each cell line had a different staining at baseline, settings were adjusted so that the percentage of each cell line staining positively for GFP-annexin V at baseline was <5%. Propidium iodide (PI) can be used to detect and exclude necrotic cells, but, because our histological analysis showed little necrosis of cells (<5-10%), PI was not used, and all GFP-staining cells were considered to be apoptotic [early apoptotic (PI negative) plus late (PI positive)].

Immunocytochemistry for Bcl-2 expression. Cells were plated on eight-chamber slides overnight for staining for Bcl-2 expression. After the cells were fixed with 50% methanol-50% acetone for 2 min and blocked with BSA (2%) for 15 min, anti-human Bcl-2 (1:200 in 2% BSA; DAKO, Carpinteria, CA) was added for 30 min. The antibody was detected with a peroxidase-labeled streptavidin-biotin detection kit (DAKO LSAB2), following instructions. In additional experiments, to detect Bcl-2 in mitotic cells, cells were first arrested in mitosis with Colcemid (20 ng/ml; Sigma) and vincristine (1 µg/ml; Sigma) for 4 h before immunocytochemical staining for Bcl-2 expression.

Immunoblotting for Bcl-2 and Bax expression. Cells, both adherent and floating, were rinsed in ice-cold PBS and then lysed in RIPA buffer (10 mM Na2HPO4, 1% Triton X-100, 10% SDS, and 0.5% deoxycholic acid) at 4°C for 1 h. Normal-appearing lung tissues obtained from two patients undergoing resection for non-small cell lung cancer (gift of Herbert Oie, National Cancer Institute, Bethesda, MD) were used as normal controls and were lysed in SDS sample buffer (60 mM Tris · HCl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromphenol blue). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Samples (30 µg) were boiled for 5 min in SDS sample buffer and then electrophoresed on 1.5-mm polyacrylamide gels. Proteins were transferred to Immobilon P-Millipore membranes along with prestained molecular-weight markers at 0.2 A overnight. After being blocked with dry milk (3%) for 3 h, membranes were incubated with the primary antibody, either mouse anti-human Bcl-2 (1:50; DAKO) or rabbit polyclonal anti-human Bax (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), for 1 h at room temperature. After being washed in PBS-Tween 0.1%, membranes were incubated with horseradish peroxidase-conjugated secondary antibody [anti-mouse IgG (1:1,000; DAKO) for Bcl-2 and anti-rabbit IgG (1:1,000; DAKO) for Bax] for 45 min followed by chemiluminescent detection (ECL Kit; Amersham).

The lysate from HL-60/Bcl-2 transfected cells (31) cultured in the RPMI medium supplemented with 500 µg/ml of G-418 (Geneticin; GIBCO) was used as the positive control for the Bcl-2 and Bax immunoblotting experiments.

Statistics. Data are expressed as means ± SE. Statistical differences among groups were determined by analysis of variance with Tukey's test to identify where the difference lay (16). A P value < 0.05 was accepted as significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Analysis of apoptosis. Mesothelioma cell lines were significantly more resistant to apoptosis than were primary cultured rabbit and human mesothelial cells, whether analyzed by acridine orange staining (Fig. 1) or by surface expression of phosphatidylserine (Table 1). There was no difference in resistance between the line with wild-type p53 (HuT 28) and those with mutant p53 (LRK1A, REN). The mesothelioma cell lines were more resistant to apoptosis due to oxidant stimuli, asbestos (10-20 µg/cm2), and H2O2 (100 µM), as well as to a nonoxidant stimulus, calcium ionophore (0.5 µM).


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Fig. 1.   Histological analysis of the resistance of mesothelioma-derived cell lines to various apoptotic stimuli. Mesothelioma lines and 1° cultured mesothelial cells were treated with crocidolite asbestos (20 µg/cm2), H2O2 (30 µM), or calcium ionophore (0.5 µM) for 18 h, harvested, and stained with acridine orange (10 µg/ml). Apoptosis was quantified by detection of condensed nuclei by fluorescence microscopy. Values are means ± SE of 4 experiments in each group. * Significant difference from apoptosis of both 1° cultured rabbit and human mesothelial cells, P < 0.05.

                              
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Table 1.   Apoptosis of mesothelioma lines and primary cultured mesothelial cells as measured by staining with GFP-annexin V

Mesothelial cell lines previously shown to have a different sensitivity to asbestos toxicity also were more resistant to asbestos-induced apoptosis than were primary cultured mesothelial cells (Fig. 2). However, the line derived from exposure to asbestos (P19A/T) did not show more resistance to asbestos-induced apoptosis than lines derived without exposure to asbestos (MEJ-T10, MeT-34, and MeT-5A).


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Fig. 2.   Resistance of asbestos- and non-asbestos-exposed mesothelial cell lines to asbestos-induced apoptosis. Mesothelial cell lines previously exposed to asbestos (P19A/T) or not exposed to asbestos (MEJ-T10, MeT-34, and MeT-5A) as well as 1° cultured mesothelial cells were exposed to crocidolite asbestos (10 or 20 µg/cm2) for 18 h, harvested, and stained with acridine orange. Data for MEJ-T10 and MeT-34 are combined. Apoptosis was quantified by detection of condensed nuclei by fluorescence microscopy. Values are means ± SE of 3 experiments in each group. * Significant difference of asbestos-induced apoptosis compared with that of both 1° cultured rabbit and human mesothelial cells, P < 0.05.

Immunocytochemistry. Only one of the three mesothelioma cell lines (HuT 28) and the transformed cell line MeT-5A stained positively for Bcl-2 (Fig. 3). Interestingly, Bcl-2 staining was only found associated with chromosomes of mitotic cells, a pattern previously described in epithelial cell lines and carcinoma cell lines (24, 44). Mesothelioma lines were then arrested in mitosis to increase the likelihood of detecting Bcl-2 in mitotic cells, but no additional Bcl-2 positive lines were found.


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Fig. 3.   Expression of Bcl-2 by immunocytochemical staining. Cells were grown on 8-chamber slides overnight, fixed, blocked with BSA, and stained with anti-human Bcl-2 (1:200 in 2% BSA) with detection by peroxidase-labeled streptavidin-biotin. The mesothelioma line HuT 28 and the immortalized mesothelial line MeT-5A expressed Bcl-2 in the nuclei of mitotic cells (arrows). LRK1A represents the other lines that were negative. Negative control, HuT 28 cells stained similarly but with omission of the primary antibody. Bar = 8 µm.

Immunoblots. Only 3 of the 14 mesothelioma cell lines stained positively for Bcl-2 but at a similar level of expression as found in normal lung tissue (Fig. 4). Interestingly, all 14 cell lines stained for the proapoptotic Bax at a level equal or greater to that in normal tissues (Fig. 5).


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Fig. 4.   Immunoblot for Bcl-2 expression in lysates of mesothelioma lines studied for their apoptotic responses (A) and in lysates from additional mesothelioma lines (B). Cells or normal lung tissues were lysed. Equal amounts of protein (~30 µg) were electrophoresed, transferred to Immobilon, blocked with dry milk (3%) for 3 h, and incubated with mouse anti-human Bcl-2 (1:50) for 1 h at room temperature. Bcl-2 was detected after incubation with horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG, 1:1,000) for 45 min followed by chemiluminescent detection. Lysate from HL-60/Bcl-2 transfected cells was used as the positive control. Bracket identifies cell lines with wild-type p53 (27). Nos. on left, molecular mass (in kDa).


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Fig. 5.   Immunoblot for Bax expression in lysates from mesothelioma lines studied for their apoptotic responses (A) and in lysates from additional mesothelioma lines (B). Immunoblot was prepared as for Bcl-2 (see Fig. 4) with the exception that the primary antibody was rabbit anti-human Bax (1:1,000) and the secondary antibody was anti-rabbit IgG (1:1,000). Bracket identifies cell lines with wild-type p53 (27). Nos. on left, molecular mass (in kDa).

    DISCUSSION
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Once primarily a study of cell proliferation, the consideration of oncogenesis has expanded to include the study of cell death. Apoptosis, an active process of regulating cell death, relies on a complex cellular program that can be subverted (32). Because apoptosis may function to delete cells with DNA damage, a failure in the apoptotic response may contribute to the formation of tumors (41, 43). Because a failure in the apoptotic response may also underlie the resistance of tumor cells to chemotherapy and radiotherapy (23), apoptosis becomes increasingly relevant to the study of cancer.

We investigated the resistance to apoptosis in mesothelioma, a tumor noted for its insensitivity to therapy (4, 34). In this study, we have found that mesothelioma cell lines are resistant to apoptosis induced by diverse stimuli. Because mesothelioma cells were resistant to both oxidant [asbestos (8), H2O2] and nonoxidant (calcium ionophore) stimuli, the block to apoptosis could exist either at multiple sites specific for the different stimuli or at a common downstream location (14).

Neither the tumor cells nor the mesothelial cell lines exhibited a selective resistance to asbestos. In a previous study, it had been shown that mesothelioma cells were selectively resistant to asbestos-induced cytotoxicity (15). In addition, cell lines derived from asbestos exposure demonstrated a greater resistance to asbestos-induced cytotoxicity than did lines without prior asbestos exposure, suggesting that exposure to asbestos had selected for resistant cells. When we looked at the same mesothelial cell lines as well as at mesothelioma-derived cell lines, we did not find this to be true for apoptosis, although the conditions of the experiments such as duration of exposure, asbestos dose, and cell density were clearly different. Although in both studies we used the same cell lines, alterations necessary to produce a cell line may have altered apoptosis specifically, leaving other responses to asbestos intact. Nonetheless, under the conditions of these experiments, the resistance to apoptosis was broad based and not specific to asbestos.

The mechanism of resistance of these mesothelioma-derived cell lines, and presumably of the original tumor itself, to apoptosis does not derive from the anti-apoptotic protein Bcl-2. Expression of Bcl-2 was found in only 3 of the 14 lysates and in only 1 of the 3 mesothelioma lines. In this mesothelioma line, HuT 28, Bcl-2 appeared only in mitosis in association with chromosomes, a pattern that may stabilize cells during the period of cell division (24, 44). This mitotic expression was not sufficient, however, to allow the detection of Bcl-2 by immunoblot, even when the cells were arrested in mitosis before immunoblotting (data not shown). As mentioned above, derivation of a cell line may specifically select for a high resistance to apoptosis, which may not be representative of the original tumor. Nevertheless, Bcl-2 overexpression is not the explanation for the resistance of these mesothelioma-derived cell lines to apoptosis. Together with the histological report of a lack of Bcl-2 in most mesotheliomas (9, 39), our data suggest that the oncogenic effects of Bcl-2 are not important in mesothelioma.

Interestingly, we found the proapoptotic protein Bax in every mesothelioma cell line. Bax has been shown to accelerate apoptosis, and Bcl-2 is thought to exert its antiapoptotic function by heterodimerizing with Bax (33, 46). Therefore, it is the ratio of Bcl-2 to Bax that appears to direct the cell away from or toward apoptosis (33, 38). The ratio in our cell lines, because of the absence of Bcl-2 in most of them, is low, with Bax mostly unopposed by the modulating effect of Bcl-2. Because p53 acts as a negative regulator of Bcl-2 and positive regulator of Bax (28), the low ratio of Bcl-2 to Bax may be a consequence of the wild-type p53 present in most of the cell lines. Nevertheless, a low Bcl-2-to-Bax ratio was also found in mesothelioma cells with mutant p53, indicating that p53 is not the only factor controlling Bcl-2/Bax levels. Indeed, in other studies of tumor lines, high Bax levels have been found in cells with mutant p53 (12), and non-p53 stimuli have been reported to enhance expression of Bax (20). Whatever the influences leading to the levels in our cells, the Bax, if functional, would be expected to shift the cellular response toward apoptosis.

Indeed, in most studies, a low ratio of Bcl-2 to Bax is associated with a favorable histological grade and responsiveness to treatment of tumors or of tumor-derived cell lines. In cells derived from testicular cancer, the relative sensitivity to apoptosis was higher than that of cells derived from bladder cancer, a difference that correlated with a lower Bcl-2-to-Bax ratio in the testicular cancer cells (11). In chronic lymphocytic leukemia, a lower ratio of Bcl-2 to Bax was found in leukemic cells from patients with a slower progression of disease (1). In neuroendocrine lung tumors, low Bcl-2-to-Bax ratios (<1) correlated with low-grade histology and better outcome (6); in urinary bladder cancers, a low Bcl-2-to-Bax ratio (<1) correlated with a lack of relapse after surgery. It is thus unusual to find a low Bcl-2-to-Bax ratio in a resistant tumor.

The presence of a proapoptotic pattern of wild-type p53 in most of the mesothelioma-derived lines together with a low Bcl-2-to-Bax ratio in all mesothelioma-derived lines suggests that the defect in both apoptosis and responsiveness to treatment lies elsewhere. Within the current and incomplete understanding of apoptotic regulation, some possibilities can be considered. A defect could arise from Bax mutations, rendering the protein nonfunctional and blocking its proapoptotic effect (26, 35), or from antagonists to Bax as seen with certain viral proteins (18). A defect could arise from abnormalities in other members of the Bcl-2 family such as proapoptotic Bad (45) and Bak (10) or anti-apoptotic Bcl-xl (3) or Mcl-1 (37). The defect in apoptosis could also lie further downstream in the part of the apoptotic pathway activated by Bax (32). Alternatively, apoptosis could be inhibited by other antiapoptotic influences, such as by growth factor stimulation or integrin activation (7).

In conclusion, we report that mesothelioma-derived cell lines exhibit a general and high level of resistance to apoptosis. Specific resistance to asbestos was not found. We have also shown that Bcl-2 is not overexpressed in mesothelioma-derived cell lines and does not account for their resistance to apoptosis. Bax, a proapoptotic protein, was detected in all lines, suggesting that the explanation for the resistance to apoptosis in mesothelioma may lie in mechanisms that counteract the function of Bax.

    ACKNOWLEDGEMENTS

We thank Dr. Steven M. Albelda (University of Pennsylvania, Philadelphia, PA) for providing mesothelioma lines, Herbert Oie (National Cancer Institute, Bethesda, MD) for providing normal lung tissue, and Dr. Louie Naumovski (Stanford University School of Medicine, Stanford, CA) for providing Bcl-2-overexpressing HL-60 cells.

    FOOTNOTES

S. R. Narasimhan and V. C. Broaddus were supported by National Institute of Environmental Health Sciences Grants ES-06331 and ES-08985 and by an Academic Senate Award.

Address for reprint requests: V. C. Broaddus, Lung Biology Center, Box 0854, Univ. of California, San Francisco, CA 94143-0854.

Received 2 July 1997; accepted in final form 29 March 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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