Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines

Lung Cancer 23 (1999) 115 – 127

Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines Patrick P. Koty, Haifan Zhang, Mark L. Levitt * Lung Cancer Program, Allegheny Uni6ersity of the Health Sciences, 320 E. North A6enue, Pittsburgh, PA 15212, USA Received 6 June 1998; received in revised form 3 November 1998; accepted 13 November 1998

Abstract Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Programmed cell death; Antisense bcl-2; Non-small cell lung cancer

* Corresponding author. Tel.: +1-412-359-8931; fax: + 1412-359-6654. E-mail address: [email protected] (M.L. Levitt)

1. Introduction More than 160 000 people in the United States died as a result of lung cancer in 1998, making it the leading cause of cancer-related deaths [1].

0169-5002/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 9 - 5 0 0 2 ( 9 8 ) 0 0 0 9 7 - X

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Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for more than 75% of all lung cancer cases. The outlook for patients with NSCLC is dismal, with only slightly more than 10% of patients alive 5 years after diagnosis [2,3]. The ineffectiveness of current preventive and treatment strategies necessitates the investigation of other means of intervention. As cancer is a disease of altered cellular homeostasis, programmed cell death (PCD) has gained increasing recognition in the past several years as a physiologic mechanism that may be relevant to cancer treatment strategies [4]. PCD is aberrantly regulated in NSCLC, rendering cells resistant to chemotherapeutic agents that induce this process [3,5]. Therefore, genetic manipulation of genes that regulate PCD may restore the altered balance in these cells between proliferation and cell death. A known regulator of the PCD pathway is Bcl-2, a protein capable of suppressing cell death in a dominant negative manner when dysregulated in human cells [6,7]. The exact molecular mechanisms by which Bcl-2 inhibits PCD have not been unequivocally established. Studies have shown that Bcl-2 inactivates inducers of PCD, such as Bax, through heterodimerization and that it is the ratio of Bcl-2, or other functionally similar genes, to the expression of PCD inducers that determines whether a cell will undergo PCD [8,9]. More recently, it has been shown that cellular localization may be a determining factor, rather than strictly the level of Bcl-2 expression [10,11]. In addition, reports indicate that Bcl-2 must be phosphorylated to function as an anti-apoptotic agent [12]. It has also been reported that Bcl-2 is capable of forming membrane channels and can disrupt the channel forming ability of Bax [13,14]. Bcl-2 has also been shown to regulate the intracellular location of Ca2 + stores [15 – 19] and to prevent cytochrome c release from the outer mitochondrial membrane to the cytosol where cytochrome c activates PCD caspases [20,21]. Furthermore, studies have indicated Bcl-2 can protect cells from death by acting as an antioxidant [22 – 25]. Although the mechanism(s) by which bcl-2 prevents PCD remain unclear, its manipulation may provide a means of restoring homeostasis and chemotherapeutic sensitivity in NSCLC.

Therapeutic strategies for those diseases overexpressing bcl-2 have focused on decreasing Bcl-2 levels. Treatment of non-Hodgkin’s lymphoma, lymphocytic leukemia, prostate and breast cancer cell lines in vitro with antisense bcl-2 vectors or oligonucleotides lowered the amounts of Bcl-2, increasing the sensitivity of these cells to chemotherapeutic agents [26–29]. In fact, antisense bcl-2 therapy has recently been conducted on patients with non-Hodgkin’s lymphoma resulting in some initial success [30]. This approach has recently been successful in reducing cell viability in several small cell lung cancer (SCLC) cell lines after treatment with antisense bcl-2 oligonucleotides [31]. This suggests a similar reduction may be observed in NSCLC; however, differences in bcl-2 expression and chemosensitivity between SCLC and NSCLC require investigating the response of NSCLC to antisense bcl-2 treatment. A much higher percentage of SCLC tumors and cell lines [32,33] than NSCLC [34–37] express elevated levels of bcl-2. Therefore, Bcl-2 may or may not be a primary inhibitor of PCD in NSCLC. Furthermore, SCLC is more chemosensitive to agents that induce PCD than is NSCLC [38–41]. Therefore, it may be easier to manipulate PCD by antisense bcl-2 treatment in SCLC if Bcl-2 is the primary PCD regulator, as suggested by the high percentage of SCLC cases that overexpress bcl-2 as compared with NSCLC. However, NSCLC cells do express elevated levels of Bcl-2, which may result in bypassing the normal PCD pathway and which may be responsible, in part, for their chemotherapeutic resistance [42,35–37]. Thus, counteracting this abnormal expression may re-establish the normal cellular response to maintain homeostasis. In fact, one study showed a decrease in cellular proliferation of NSCLC cell lines following antisense bcl-2 treatment [43]. However, this study did not indicate whether antisense bcl-2 treatment resulted in decreased Bcl-2 protein levels, nor did it determine whether the decrease in cellular proliferation was due to PCD. The present study represents the first in a series of preliminary studies leading up to attempted gene therapy for NSCLC employing antisense bcl-2 treatment. Herein we establish that bcl-2 and bax are expressed in several NSCLC cell lines

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and that these cell lines have a functional endogenous PCD pathway. We then demonstrate the PCD-inducing, antiproliferative effect of antisense bcl-2 treatment on these cell lines. In this study, we use three morphologically distinct NSCLC cell lines that we have previously characterized: NCIH226 (squamous), NCI-H358 (adenocarcinoma), NCI-H596 (adenosquamous) [44 – 46].

2. Material and methods

2.1. Cell lines The NSCLC cell lines NCI-H226 (squamous), NCI-H358 (adenocarcinoma), NCI-H596 (adenosquamous) were cultured in RPMI-1640 (supplemented with 0.3 mg/ml l-glutamine, 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 mg/ml streptomycin, 0.5 mg/ml fungizone) (Gibco/BRL) at 37°C in an atmosphere of 5% CO2. Cells were plated and harvested either in log phase or confluent growth for the mRNA quantitation, in confluent growth for immunohistochemical staining analysis, or grown to 80% confluence for the synthetic antisense bcl-2 oligonucleotide study.

2.2. Re6erse transcription-polymerase chain reaction This procedure involved isolation of total mRNA (Invitrogen) from cells in log phase or confluent growth. First strand cDNA was synthesized using 0.5–2.5 mg total polyadenylated mRNA template, 0.5 mg oligo-dT15 primer (Boehringer Mannheim) and sterile H2O to a final volume of 10.5 ml, then denatured at 70°C for 2 min and placed on ice for 2 min. Fifteen U AMV reverse transcriptase (Boehringer Mannheim), 10 mM each dNTP, and 20 U RNase Inhibitor (Boehringer Mannheim) were added to a final volume of 20 ml, incubated at 42°C for 1 h, then heated for 2 min at 94°C. The reaction mixture was then diluted to a final volume of 100 ml with sterile H2O.

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2.3. Polymerase chain reaction amplification Polymerase chain reaction (PCR) reaction mixtures contained 1 ml diluted reverse transcription-PCR (RT-PCR) product, 1× PCR buffer (Perkin Elmer); 2.5 mM each dATP, dTTP, dGTP, 0.625 mM cold dCTP, and 0.5 mCi [a-32P] dCTP, 20 pmol unique forward and reverse primer, 0.3 pmole b-actin forward and reverse primers, and 0.5 U AmpliTaq polymerase (Perkin Elmer) in a final volume of 5 ml. DNA thermal cycler (Perkin Elmer) conditions were (94°C, 1 min; 61°C, 1 min; 72°C, 1 min), 35 cycles. We designed the following unique primers for PCR reactions: bcl-2 (448 bp amplified product) ( forward 5%-ccctccagatagctcatt, re6erse 5%-ctagacagacaaggaaag); bax (579 bp amplified product) ( forward 5%-atggacgggtccggggag, re6erse 5%-tcagcccatcttcttcca); and b-actin (382 bp amplified product) ( forward 5%-tctacaatgagctgcgtg, re6erse 5%-ccttaatgtcacgcacga). The primers were synthesized by MGB Research Support Facilities (University of Pittsburgh). The PCR products were electrophoresed on a 3% agarose gel in 1× TBE buffer and vacuum dried onto 3M Whatman filter paper.

2.4. mRNA semi-quantitation The radioactively labeled PCR products dried onto 3M Whatman filter paper were semi-quantified using a Betagen Betascope 603 (Tritech Field Engineering). Variations in template cDNA concentrations were normalized by co-amplification with b-actin using the following formula: (X cpm Y cell line) (b− actin cpm Y cell line/b− actin cpm Z cell line) = normalized X cpm Y cell line

where X is the cpm for either bcl-2 or bax for the Y cell line, Y is the NCI-H226, NCI-H358, or NCI-H596 cell line, and Z is the constant cell line used to normalize all other cell lines. Variations in radioisotope activity between experiments were normalized to the total b-actin cpm after normalizing for concentration variations as follows:

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(Total cpm b −actin Experiment 1 for gene A) (Total cpm b −actin Experiment 2 for gene A) = Normalizing factor Experiments were done in duplicate and normalized values were averaged.

2.5. Immunohistochemical staining for DNA fragmentation A terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) kit (Oncor) was used to detect 3%-OH DNA strand breaks. Isolated cells were fixed in 1% formalin for 10 min to maintain cell morphology. Cells (1×106) were put on a microslide to dry at room temperature overnight. After the cells were fixed in 10% neutral buffered formalin in PBS, pH 7.4, for 10 min and then in cold acetone for 10 min, the slides were washed once with TdT buffer (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate) and then incubated with digoxigenin-conjugated dUTP and TdT at 37°C for 90 min. After incubation with peroxidase conjugated antidigoxigenin antibodies, bound peroxidase was detected using the substrate 3-amino-9-ethyl carbazole (AEC). The percentage of cells staining positively was obtained by counting 200 cells per field, from a minimum of three fields using a 10× eyepiece and 20× objective.

2.6. Dual-staining for DNA fragmentation and Bcl-2 Cells were treated as before for TUNEL staining followed by incubation with monoclonal antibody for Bcl-2 (clone 124; DAKO) for 120 min at room temperature. After washing with PBS, biotinylated horse anti-mouse antibodies (Vector Laboratories) were added for 60 min followed by the addition of alkaline phosphatase-conjugated streptavidin (Vector Laboratories) for 60 min. Bound alkaline phosphatase was detected with alkaline phosphatase substrate III (Vector Laboratories).

2.7. Synthetic oligonucleotide treatment We designed a 20 bp oligonucleotide that was specific for the antisense strand of bcl-2, as verified by a GenBank database search. We also designed a 20 bp complimentary sense strand to the antisense oligonucleotide for bcl-2 for the competitive assay. Additionally, as a control for specificity and toxicity, we designed a 20 bp nonsense oligonucleotide, which showed less than 85% homology to any sequence within the GenBank database. Oligonucleotide sequences are as follows: nonsense oligonucleotide 5%gtatgacctagcggttgt-3%, antisense bcl-2 oligonucleotide 5%-gttatcgtaccctgttctcc-3%, sense bcl-2 5%-ggagaacagggtacgataac-3% (all oligonucleotides were synthesized using phosphorothioate nucleotides by Research Genetics). Cells were grown to  80% confluence. Subsequently, the cells were washed once with serum-free RPMI-1640 media, overlaid with the appropriate solution (serumfree media control, 0.3 mM nonsense oligonucleotide in serum-free media, 0.3 mM antisense bcl-2 oligonucleotide in serum-free media, 0.3 mM antisense bcl-2/0.3 mM sense bcl-2 in serumfree media, or 0.3 mM antisense bcl-2/3.0 mM sense bcl-2 in serum-free media) and analyzed at 3, 6, 12, and 24 h.

2.8. Enzyme-linked immunosorbent-based assay for oligonucleosomal DNA fragmentation Oligonucleosomal DNA fragmentation, a marker of PCD, in the treated NSCLC cell lines was quantified using a 96-well plate enzymelinked immunosorbent-based assay (Boehringer Mannheim). The detection process first involves lysis of cell plasma membranes, followed by isolation of histones using anti-histone antibodies, then labeling histone/DNA complexes with an anti-DNA-peroxidase conjugated antibody. Finally, the bound immunocomplexes labeled with peroxidase are visualized using 2,2%-azino-di-[3ethylbenzthiazoline sulfonate] as a substrate. This reaction was quantitated photometrically by measuring absorbance at 405 nm.

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2.9. Statistical analysis Statistical significance between two observations was compared using a two-tailed Student’s t-test. However, when three parameters were evaluated, we used an analysis of variance procedure for primary analysis followed by a posthoc multiple mean test, Tukey’s HSD test. A P value of less than 0.05 and a power of 80% was accepted as statistically significant. The software package Systat 6 for Windows was used (SPS, 1996). All experiments were done in duplicate.

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In general, it appeared that bcl-2 is upregulated during confluent growth compared with log phase in all three NSCLC cell lines (NCIH226 and NCI-H358 statistically significant difference) (Fig. 1A). Although not significant, Bax appeared to be upregulated during confluent growth in NCI-H358 and NCI-H596 cell lines but was unchanged in the NCI-H226 cell line (Fig. 1B). There was, however, a dramatic change in the bcl-2:bax ratio from confluent to log phase growth in the NCI-H226 cell line, whereas the ratios did not change in NCI-H358 and NCI-H596 (Fig. 1C).

3. Results

3.2. Spontaneous DNA fragmentation 3.1. Expression le6els of bcl-2 and bax mRNA in NSCLC cells We first determined if bcl-2 and bax were expressed endogenously in NCI-H226, NCI-H358, and NCI-H596 cells. A comparison of these levels was performed on cells in log phase and confluent growth, since the need to regulate PCD may vary under different growth conditions. Therefore, if bcl-2 and bax are involved in regulating PCD in NSCLC, changes in their expression levels may be expected under different growth conditions. Determination of mRNA levels involved isolation of mRNA, first strand cDNA synthesis by RT-PCR with an oligo-dT15 primer, and PCR amplification using unique primer sets for bcl-2 and bax. Variations in template cDNA concentrations and radioisotope activity were normalized by co-amplification with b-actin. This approach is only semi-quantitative, since different sized products with different base pair compositions are amplified. Thus, it is not possible to make an absolute quantitation of the message of interest. However, it is possible to evaluate changes in the level of a particular message under different conditions and between different cell lines for the same message. In addition, it is not possible to determine the actual molar ratio of bcl-2 to bax, and therefore which message (bcl-2 or bax) is in excess of the other. However, it is possible to detect changes in the ratio of their PCR products.

All three cell lines underwent similar low levels of spontaneous PCD during confluent growth, as determined by in situ immunohistochemical staining for DNA fragmentation (Fig. 2). In contrast, DNA fragmentation is not detectable in cells during log phase growth (data not shown). That these NSCLC cells express both bcl-2 and bax and are capable of utilizing a PCD pathway characterized by DNA fragmentation suggests that bcl-2 and bax may be regulators of the PCD pathway in NSCLC. By using the more sensitive TUNEL staining technique, we were able to detect spontaneous DNA fragmentation that we were previously unable to detect by agarose gel electrophoresis [45].

3.3. Antisense bcl-2 treatment decreases proliferation We next investigated whether decreasing the expression of bcl-2 would increase the amount of spontaneous PCD in these three morphologically distinct NSCLC cell lines. We did so by designing a synthetic bcl-2 antisense oligonucleotide using phosphorothioate nucleotides. We designed the antisense primer to hybridize near the start codon of the mRNA strand. A nonsense oligonucleotide was designed in a similar fashion and was not homologous to any other sequence within the GenBank database. This

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nonsense primer was used as a control for cytotoxicity and possible nonspecificity of the synthetic phosphorothioate labeled oligonucleotide. Treatment with the antisense bcl-2 oligonucleotide resulted in reduced cell proliferation in all three cell lines, as measured by counting the total number of viable and nonviable cells (Fig. 3). The data in Fig. 3 represents cell counts at 24 h. Similar results were obtained at 3, 6, and 12 h for all three cell lines.

3.4. Viability decreased after treatment with antisense bcl-2 To determine whether the decrease in cell number was due to a loss in viability as opposed to a slowing of the cell cycle, we determined cell viability by a trypan blue dye exclusion assay. The addition of trypan blue dye to the treated and untreated cells revealed that antisense bcl-2 treatment resulted in decreased cell viability, evidenced

Fig. 1. Expression of bcl-2 and bax in NSCLC cell lines. mRNA was isolated from cells in log phase or confluent growth, and RT-PCR was performed for first strand cDNA synthesis and amplification. Selected genes were quantified by PCR analysis. The morphologically distinct cell lines analyzed were NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Quantitation was based on the amount of radiolabeled nucleotide incorporated into a PCR product measured as counts per minute. (A) bcl-2 mRNA levels for cells in log phase and confluent growth, (B) bax mRNA levels for cells in log phase and confluent growth, (C) bcl-2 and bax mRNA levels in cells in confluence vs log phase growth. *, Statistically significant difference, P 50.05. Experiments were performed in duplicate.

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Fig. 2. Spontaneous levels of PCD in NSCLC cell lines. Amount of spontaneous PCD as measured by DNA fragmentation in the NSCLC cell lines NCI-H226, NCI-H358, and NCI-H596 grown to confluence as measured by immunohistochemical staining. Experiments were performed in triplicate.

by an increase in the number of trypan blue permeable cells in all three cell lines (Fig. 4). The data in Fig. 4 represent the number of trypan blue positive cells at 24 h. Similar results

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Fig. 4. Cell viability in NSCLC cells treated with antisense bcl-2. An increase in nonviable cells, as measured by the trypan blue assay, was observed in all three cell lines when treated with antisense bcl-2 for 24 h, as compared with controls. Similar results were obtained at 3, 6, and 12 h. *, Statistically significant difference, P 50.05. Experiments were performed in triplicate.

were obtained at 3, 6, and 12 h for all three cell lines.

3.5. Increased DNA fragmentation following treatment with antisense bcl-2

Fig. 3. Cell proliferation in NSCLC cells treated with antisense bcl-2. Proliferation was measured by total cell count, as determined using a hemacytometer under light microscopy. In all three cell lines, when compared with controls, treatment with antisense bcl-2 for 24 h caused a decrease in cell proliferation as measured by total cell counts. Similar results were obtained at 3, 6, and 12 h. This data is representative of three separate experiments.

To determine if the decrease in cell viability was due to PCD and not necrosis, we measured the amount of DNA fragmentation (a marker of one PCD pathway) occurring in the treated and untreated NSCLC cells. The increase in DNA fragmentation in all three cells lines indicated that the decrease in cell viability caused by antisense bcl-2 treatment was the result, at least in part, of an increase in spontaneous PCD rather than necrosis (Fig. 5). Antisense bcl-2 treatment seemed to have the most dramatic effect on DNA fragmentation in cell lines with squamous features (NCI-H226 and NCI-H596) and a less dramatic effect in the adenocarcinoma cell line (NCI-H358). For these experiments, DNA fragmentation was measured by an enzyme-linked immunosorbent-based assay. Similar results were obtained by immunohistochemical staining using the TUNEL method to detect DNA fragmentation (Fig. 6, red color).

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3.6. Antisense bcl-2 treatment of NSCLC cells specifically increases DNA fragmentation To ensure that the increase in spontaneous DNA fragmentation was specifically due to bcl-2 downregulation, we co-transferred the antisense bcl-2 and its complimentary sense strand bcl-2 oligonucleotides. The presence of the sense strand bcl-2 prevented the increase in DNA fragmentation observed by antisense bcl-2 treatment alone, thus indicating the specificity of the antisense bcl-2 treatment (Fig. 7). Although not quantified,

immunohistochemical staining confirmed specificity by decreasing the amount of Bcl-2 (blue color) in antisense treated cells as compared with media only and nonsense treated controls (Fig. 6)

4. Discussion As cancer is a disease of altered cellular homeostasis, recent strategies have targeted the induction of PCD as an attractive paradigm for the treatment of this disease [4]. Our data have

Fig. 5. PCD in NSCLC cells treated with antisense bcl-2. (A) NCI-H226, (B) NCI-H358, (C) NCI-H596. DNA fragmentation, as measured by an enzyme-linked immunosorbent-based assay, was used to quantify programmed cell death. Treatment of the cells with antisense bcl-2 resulted in an increase in spontaneous programmed cell death, as compared with controls. *, Statistically significant difference, P50.05. Experiments were performed in duplicate.

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Fig. 6. Dual immunohistochemical staining of NCI-H226 cells for DNA fragmentation and Bcl-2 expression. Cells grown to 80% confluence were overlaid for 24 h with serum free media (A) alone, or containing (B) 0.3 mM nonsense oligonucleotide or (C) 0.3 mM antisense bcl-2 oligonucleotide. The cells were fixed and stained for DNA fragmentation using the TUNEL method (red color) and for Bcl-2 expression (blue color). Treatment with antisense bcl-2 oligonucleotide resulted in decreased expression of Bcl-2 and increased DNA fragmentation. Experiments were performed in duplicate. Magnification, ×200.

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Fig. 7. Specificity of antisense bcl-2 oligonucleotide inhibition. (A) NCI-H226, (B) NCI-H358, (C) NCI-H596. DNA fragmentation, as measured by an enzyme-linked immunosorbent-based assay, was used to quantify programmed cell death. Competition of sense bcl-2 strand with antisense bcl-2 reversed the increase in PCD caused by antisense bcl-2 treatment. *, Statistically significant difference, P 50.05. Experiments were performed in duplicate.

shown that both bcl-2 and bax are expressed in three morphologically distinct NSCLC cell lines and, therefore, may contribute to the regulation of PCD in these cell lines. In agreement with a previously published report [43], we have shown that decreasing the levels of bcl-2 by antisense treatment resulted in a decrease in cellular proliferation. In addition to documenting a decrease in Bcl-2 protein levels upon antisense bcl-2 treatment, we have shown in the present study that the decrease in cellular proliferation is due to PCD. The reversal of the PCD-inducing effects of anti-

sense bcl-2 by co-treatment with the sense strand of bcl-2 confirmed the specificity of antisense bcl-2 action. Taken together, our results indicate that all morphological types of NSCLC can be genetically manipulated to spontaneously enter the bcl-2 regulated PCD pathway. Our results suggest that the regulation of PCD in NSCLC may be dependent upon the proliferative state, since both bcl-2 and bax have lower expression (with the exception of bax in NCIH226, where it remains unchanged) in the log phase as compared with confluent growth. It may

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be that, as cells approach confluence and the growth rate decreases, spontaneous PCD begins to occur in response to increasing levels of PCD inducers, such as Bax. The concomitant increase in Bcl-2 levels may occur as a reaction to the increase in PCD inducers and may represent vestiges of the epithelial tendency to maintain cellular homeostasis. Our studies also suggest that the amount of endogenous Bcl-2 may influence the susceptibility of the cell to antisense bcl-2 treatment. The squamous cell lines with higher levels of bcl-2 than the adenocarcinoma cell line were more susceptible to increased DNA fragmentation via decreased bcl-2 expression. Furthermore, in terms of the bcl-2:bax ratio, in the cell lines with squamous features the ratio favors bcl-2, whereas in NCIH358 cells, the ratio favors bax. These data suggest that antisense bcl-2 treatment is more effective in cells where bcl-2 may be a more important factor in preventing DNA fragmentation. In cells where bcl-2 is already lower, other factors may be more influential in preventing cell death. It is, therefore, possible that those patients whose tumors have higher levels of bcl-2 or a bcl-2:bax ratio may be more responsive to treatment involving bcl-2 antagonism. Several studies have investigated the expression of the Bcl-2 protein in NSCLC as a prognostic indicator [35–37,42]. Paradoxically, those patients whose tumor cells express Bcl-2 have a better survival rate than those that do not express detectable levels of Bcl-2. Similar to our finding that the cell lines with squamous features, NCI-H226 (squamous) and NCI-H596 (adenosquamous) expressed higher levels of bcl-2, patients with squamous carcinoma had higher levels of bcl-2. Those patients with adenocarcinoma had higher levels of bax and lower levels of bcl-2, similar to our finding in the adenocarcinoma cell line, NCIH358. Therefore, tumors expressing bcl-2 may remain sensitive entering the PCD pathway and may be resisting death in order to accumulate further alterations to become metastatic. Tumor cells not expressing Bcl-2 may have already undergone sufficient changes that ensure cell survival in a metastatic state and no longer need Bcl-2 to resist cell death. These cells may have expressed

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Bcl-2 at an earlier time point, including during a preneoplastic state, but no longer need it for cell survival. Immunohistochemical studies performed in our laboratory on primary lung tissue have shown increased expression of Bcl-2 in preneoplastic as well as neoplastic tissue, suggesting that alterations in bcl-2 expression may be an early change in lung tumorigenesis [47]. This study represents the initial step in a series of experiments aimed at developing PCD induction through gene therapy as a treatment strategy for NSCLC. We have shown that three morphologically distinct NSCLC cell lines can be genetically manipulated to induce PCD. It is now necessary to determine whether single or multiple gene transfers will result in maximal increases in PCD and whether primary NSCLC tumor cell cultures can be manipulated in the same way. Future studies are also aimed at determining appropriate targeting methods to eliminate or minimize the potential for affecting normal tissues.

Acknowledgements Supported by the Allegheny University of the Health Sciences Cancer Center (MLL), the Allegheny General Hospital Auxiliary grant 96-0452P from the Allegheny University of the Health Sciences, and grant CA73012-01A2 from the National Cancer Institute (PPK). We would like to gratefully thank Jane Mayotte for her technical assistance, Dr Janice Sabatine for editing this manuscript and Kathi Pater for administrative assistance.

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