Therapy (2000) 7, 2051–2057  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt ACQUIRED RESEARCH ARTICLE Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small lung cell

T Saeki1, A Mhashilkar2, S Chada2, C Branch1, JA Roth1 and R Ramesh1 1Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas, MD Anderson Cancer Center; and 2Introgen Therapeutics Inc., Houston, TX, USA

The differentiation-associated gene-7 (mda-7), and Bak expression was up-regulated in wild-type cloned from a human melanoma cell line H0–1, is known to tumor cell lines, but not in p53-null cells, suggesting that induce tumor cell-selective growth inhibition in an intact p53 pathway was required for Bax and Bak induc- cells in vitro and loss of tumorigenicity . Yet, the tion. However, in all three cancer cell lines tested, activation mechanisms underlying these effects are still unknown. of the cascade and cleavage of poly(ADP-ribose) Therefore, we investigated these mechanisms on the mol- polymerase (PARP) appeared to be independent of the p53 ecular level in human non-small cell lung carcinoma mutational status. Together, these results suggest that (NSCLC) cells in vitro. Overexpression of mda-7 protein by may be induced via multiple pathways by Ad-mda- Ad-mda-7 significantly suppressed proliferation and induced 7 in cells and that Ad-mda-7 has the potential to G2/M cell cycle arrest in wild-type p53 (A549, H460), and become a novel therapeutic for clinical cancer gene therapy. p53-null (H1299) non-small cell lung cancer cell lines, but Gene Therapy (2000) 7, 2051–2057. not in normal human lung fibroblast (NHLF) cells. p53, Bax,

Keywords: mda-7; gene therapy; apoptosis; lung cancer; caspase

Introduction of tumor formation of MCF-7 xenograft following mda- 7 expression. However, when mda-7 was overexpressed Recently, the melanoma differentiation-associated gene-7 in normal human cells (human mammary epithelial cells (mda-7) has gained attention as a potential tumor sup- and HBL-100), only limited cytotoxic effects were seen. pressor. Its cDNA was originally isolated by subtraction In addition, Bax protein expression was up-regulated fol- hybridization from a human melanoma cell line (H0–1) lowing overexpression of mda-7 in breast cancer cell lines that was induced to differentiate terminally by treatment T47D and MCF-7. This implied that the Bax protein may with recombinant human fibroblast interferon-beta (IFN- be involved in the apoptotic pathway induced by mda- ␤) plus mezerein (MEZ). The mda-7 gene encodes a pro- 7, an especially interesting hypothesis since Bax and tein of 206 amino acids with a predicted molecular mass other Bcl-2 protein family are known to be key of 23.8 kDa.1 Several other melanoma differentiation- regulators of apoptosis and important determinants of associated genes have also been isolated using this tech- 6 nique, including the cyclin-dependent kinase inhibitor cell fate. Previous studies have defined mda-7 as a novel p21 (mda-6)2 and mda-9.3 mda-7 mRNA levels are higher having an antitumor activity in in actively proliferating normal melanocytes than in vitro and suggested that it inhibits a diverse number of melanoma cells, and the level of mda-7 expression is . However, the functions of the mda-7 gene and inversely related to melanoma progression,1 suggesting its downstream pathways remain unknown. Conse- that mda-7 is a novel tumor suppressor gene whose quently, to understand better how mda-7 functions and expression must be inhibited for tumor progression to to help broaden the scope of current anticancer gene ther- 7–9 occur. apy strategies, we tested the antitumor effects of a Indeed, by using a colony formation assay, Jiang et al4 novel adenoviral vector carrying the mda-7 gene (Ad- found that mda-7 is a potent growth-suppression gene in mda-7) on non-small cell lung cancer lines and investi- cancer cells of diverse origin. Furthermore, Su et al5 dem- gated the underlying mechanisms of tumor killing. onstrated induction of apoptosis by overexpression of In the present study, we demonstrate Ad-mda-7 may mda-7 in breast cancer cell lines in vitro and inhibition function by triggering more than one apoptotic pathway to kill both wild-type p53-expressing (p53wt) and p53- deleted (p53null) lung cancer cell lines. Correspondence: R Ramesh, Department of Thoracic and Cardiovascular Surgery, The University of Texas, MD Anderson Cancer Center, 1515 Holcombe Boulevard, Box 23, Houston, TX 77030, USA Received 26 June 2000; accepted 30 August 2000 Ad-mda-7 in lung cancer tumor suppression T Saeki et al 2052 Results

mda-7 Expression following Ad-mda-7 infection in lung tumor and normal fibroblast cells To detect mda-7 expression in cells, human lung cancer cells (A549, H460 and H1299) and normal human fibro- blast (NHLF) cells were infected with Ad-mda-7. Forty- eight hours later, cells were fixed and stained with anti- mda-7 . Uninfected cells stained with the same antibody served as controls. High levels of mda-7 expression were observed primarily in the and perinuclei of both tumor cells and normal cells but not in the nucleus. mda-7 Expression was not observed in uninfected control cells (Figure 1).

Overexpression of mda-7 results in inhibition of cell proliferation and G2/M cell cycle arrest A549, H1299, H460 and NHLF cells were grown in six- well plates (5 × 104 per well) and infected with Ad-mda- 7 or Ad-Luc. Mock-infected (PBS) cells served as negative control. Following infection, viable cells were counted on each of days 1 to 5. Significant inhibition (P Ͻ 0.01) of cell proliferation was observed in all of the tumor cell lines infected with Ad-mda-7, as compared with control cells infected with Ad-Luc or treated with PBS. Signifi- cant suppression of cell proliferation was not observed in NHLF cells treated with Ad-Luc and Ad-mda-7 (Figure 2a). Cell cycle analysis using PI staining indicated an increase in the percentage of the G2/M population at 48 h after infection in tumor cells treated with Ad-mda- 7 as compared with tumor cells treated with PBS or Ad-Luc (Figure 2b).

Overexpression of mda-7 induces apoptosis in tumor cells but not in normal fibroblasts Following infection of Ad-mda-7, tumor cells (A549, H460 and H1299) and normal cells (NHLF) were ana- lyzed for apoptotic change by annexin V assay and Figure 2 mda-7-Induced inhibition of tumor cell proliferation and G2/M cell cycle arrest in vitro. (a) Three NSCLC cell lines and NHLF cells were TUNEL staining. Forty-eight hours after Ad-mda-7 infec- infected with Ad-mda-7 or Ad-Luc, or treated with PBS. Cell viability tion, an increase in annexin V-positive cells, an indicator was determined on each of days 1, 2, 3, 4 and 5 after infection. Tumor of initial apoptotic changes, was observed in the three cells infected with Ad-mda-7 (¼) were significantly growth suppressed (P tumor cell lines tested by FACS analysis. However, no Ͻ 0.01 on day 5) when compared with Ad-Luc-infected („) or PBS- changes were observed in cells infected with Ad-Luc or treated cells (᭜). However, NHLF cells were not growth inhibited by both mock. In contrast, normal cells infected with either Ad- Ad-Luc and Ad-mda-7 when compared with PBS treated controls. (b) Cell-cycle analysis of A549 and H1299 tumor cells at 48 h after treat- mda-7 or Ad-Luc did not demonstrate any change in the ment with Ad-mda-7 (í), Ad-Luc („), or PBS (a). G2/M arrest was number of annexin V-positive cells (Figure 3a). To con- observed in both A549 and H1299 cells infected with Ad-mda-7. firm these results further, TUNEL staining was per- formed 72 h after infection. Tumor cells but not normal cells were observed to undergo apoptosis following mda- 7 infection. No changes were observed in any of the cell lines infected with Ad-Luc (Figure 3b).

Expression of mda-7 results in up-regulation of proapoptotic p53, Bax and Bak in p53wt tumor cells Cells wild-type for p53 (A549, H460, and NHLF) and null for p53 (H1299) were infected with Ad-mda-7 and Ad- Luc. Cells were harvested at 24, 48 and 72 h after infec- tion and extracts were prepared for Western blot analy- Figure 1 Expression of mda-7 protein by Ad-mda-7. Tumor cells (A549, sis. Extracts from mock-infected cells were used as an H460 and H1299) and normal human lung fibroblast (NHLF) cells were additional control. mda-7 Protein expression was infected with Ad-mda-7, and analyzed for mda-7 expression at 48 h later by immunohistochemical staining. Uninfected cells were analyzed under detected in all of the Ad-mda-7-infected cell lines but not the same conditions. Cytoplasmic and perinuculear staining was observed in any mock-infected controls or Ad-Luc-infected cells. in infected cells. Further analysis of these cell extracts demonstrated up-

Gene Therapy Ad-mda-7 in lung cancer tumor suppression T Saeki et al 2053

Figure 3 mda-7-Induced apoptosis in tumor cells but not normal cells. (a) Tumor cells (A549, H460 and H1299) and NHLF cells were infected with Ad-mda-7 or Ad-Luc, or treated with PBS. Forty-eight hours later cells were analyzed for apoptosis by annexin V assay. In the three tumor cell lines, Ad-mda-7 infection (í) induced apparent increase of annexin V-positive cells as compared with PBS (a) and Ad-Luc („) treated controls, while no increase in positive cells was observed in NHLF cells after either vector infection. (b) Seventy-two hours after infection, cells were analyzed by TUNEL staining. Apoptotic cell death was observed in tumor cells but not in NHLF cells. Arrows indicate cells undergoing apoptotic cell death. regulation of protein levels of p53 (up to 11.0-fold over Seventy-two hours after infection cells were harvested control infected with PBS and 5.4-fold over luciferase and analyzed for annexin V, an early activator of infected control), Bax (up to 2.2-fold over PBS control and apoptosis, by flow cytometry. Apoptosis induced by Ad- 2.7-fold over luciferase), and Bak (up to 2.0-fold over PBS mda-7 was 48% inhibited by Z-VAD.FMK, while no inhi- and 2.5-fold over luciferase) in p53 wild-type A549 and bition was observed in Ad-Luc-infected cells (Figure 6). H460 tumor cells after Ad-mda-7 infection. As expected, no expression or modulation of p53 was seen in p53- Discussion deleted H1299 cells. Moreover, no change in Bax or Bak protein levels was observed in H1299 cells. In NHLF As the development of gene therapies for cancer con- cells, p53, Bax and Bak protein levels were not changed tinues, the list of novel therapeutic agents grows. Among following mda-7 expression (Figure 4). The expression these agents are those for targeting specific molecular level of Bcl-2 was not significantly changed in any of the lesions or pathways unique to tumor cells and maximiz- three tumor cell lines analyzed (data not shown). ing anticancer efficacy. Recently, one such agent, mda-7, has been shown to inhibit growth and induce apoptosis Caspase cascade activation and cleavage of PARP upon its transfer into breast cancer cells.5 The mech- following mda-7 expression anisms underlying this anticancer activity of the mda-7 Since mda-7 expression results in up-regulation of pro- gene are unknown; however, induction of apoptosis by apoptotic genes and induces apoptosis in tumor cells, the Ad-mda-7 in a p53-independent manner has been dem- activation of was studied by Western blot analy- onstrated.5 In the present study, we extended these stud- sis. These analyses demonstrated activation of a caspase ies and evaluated Ad-mda-7 as an antitumor agent in cascade including cleavage of caspase-9 and caspase-3 at lung cancer. 48 h after Ad-mda-7 infection in A549 and H460 cells and In the present study, enforced expression of mda-7 by at 72 h after infection in H1299 cells. Cleavage of PARP, Ad-mda-7 infection of lung cancer cell lines significantly a substrate for the caspases, was observed at 48 h in A549 suppressed cell proliferation by utilizing a pathway and H460 cells and at 72 h in H1299 cells. Caspase-9, cas- mediated via G2/M cell cycle arrest and resulting in pase-3 and PARP were not cleaved in NHLF cells apoptosis. It is unknown whether mda-7 induces G2/M (Figure 5). cell cycle arrest directly or indirectly via some alternative mechanisms. One possibility is that mda-7 and p21 coordi- Apoptosis induced by Ad-mda-7 was inhibited by nately affect cell cycle kinetics. This has been suggested pancaspase inhibitor by the fact that the technique used to isolate mda-7 was To determine the role of caspase in mda-7-mediated previously used to clone mda-6, which was later ident- tumor killing, H1299 cells were infected with Ad-mda-7 ified as the p21 gene from terminally differentiated H0– or Ad-Luc in the presence or absence of pancaspase 1 cells.2 Our analysis of the effects of mda-7 on normal inhibitor Z-VAD.FMK (Calbiochem, San Diego, CA, lung fibroblast cells demonstrated that Ad-mda-7 infec- USA)10,11 containing culture medium (1.0 ␮g/ml). tion did not inhibit cell growth in NHLF cells. The differ-

Gene Therapy Ad-mda-7 in lung cancer tumor suppression T Saeki et al 2054

Figure 4 Up-regulation of proapoptotic genes by mda-7 in p53wt tumor cells but not in p53null tumor cells. p53wt (A549, H460 and NHLF), and p53null (H1299) cells were infected with Ad-mda-7 or Ad-Luc, or treated with PBS. Proteins were then extracted from virus-infected and PBS-treated cells and separated by SDS-PAGE. All four cell lines infected with Ad-mda-7 strongly expressed mda-7; no expression was seen in PBS-treated or Ad-Luc- infected groups. p53, Bax and Bak protein expression was increased in Ad-mda-7-infected A549 and H460 cells as compared with other controls. In H1299 cells, no p53 protein was detected and no difference in Bax and Bak protein levels was observed in any treatment groups. In NHLF cells, up- regulation of p53, Bax and Bak protein expression was not observed. The changes in the levels of expression were determined using Image Quant software (Molecular Dynamics; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and are expressed as ratio relative to the level in PBS-treated cells, which was arbitrarily set at 1.0.

Figure 5 Mediation of apoptosis by mda-7 via caspase activation. A549, H460, H1299, and NHLF cells were infected with Ad-mda-7 or Ad-Luc. Proteins were then extracted from cells and separated by SDS-PAGE. Activation of caspase-9 and caspase-3 and cleavage of PARP, a caspase substrate, as indicated by the presence of a cleaved product, were observed at 48 h after infection with Ad-mda-7 in A549 and H460 cells and after 72 h in H1299 cells. Neither activation of caspase-9 and caspase-3 nor cleavage of PARP was detectable in any Ad-Luc-infected group. In NHLF cells activation of caspase-9 and caspase-3, or cleavage of PARP was not seen.

Gene Therapy Ad-mda-7 in lung cancer tumor suppression T Saeki et al 2055 independently of p53, Bax and Bak expression. It might be possible to say that the main apoptotic pathway by Ad-mda-7 infection is independent of the p53 pathway and that the p53-dependent pathway seen in p53wt tumor cells additionally participates in apoptotic mechanisms thereby enhancing the apoptotic effects of mda-7. Additionally, we investigated the effects of mda-7 overexpression on Bax protein-deficient and p53-mutated human prostate cancer cell line DU14517 and found that overexpression of mda-7 induced apoptosis and activated a common caspase cascade including cleavage of caspase- 9 and caspase-3. No change in p53 and Bak protein levels was seen (data not shown). Together, these results imply that the mda-7-mediated apoptotic pathway is independent of Bax status. Figure 6 Inhibition of apoptosis by pancaspase inhibitor z-VAD-FMK. Moreover, Ad-mda-7 infection on H1299 cells and Bax- H1299 cells were infected with Ad-mda-7 or Ad-Luc under the conditions deficient DU145 cells activated caspase-9, a target of cyto- of medium with or without pancaspase inhibitor z-VAD.FMK (1.0 chrome c and Apaf-1 complex and a putative initiator of ␮ g/ml). Seventy-two hours after infection, analysis of cells for apoptosis caspase activity.18 No differences were observed in apop- with annexin V assay kit demonstrated a 48% inhibition in apoptosis in totic pathways downstream of mitochondrial activation Ad-mda-7 infected cells in the presence of z-VAD.FMK. between cells that have an abnormality in either p53 status or Bax status and cells that carry the wild types of ences in toxicity to mda-7 between normal cells and these genes. However, activation of the caspase cascade tumor cells remain unknown. was detected at 48 h after infection in A549 and H460 To understand further the underlying mechanism of (p53wt) versus 72 h in H1299 (p53null) and DU145 (p53mut) tumor killing mediated by mda-7 expression, we ana- cells, suggesting that the difference in the ability of their lyzed the effects of such expression on some of the apoptotic signaling pathways to reach the point of mito- known proapoptotic genes (ie p53, Bax and Bak). Follow- chondrial release of cytochrome c may be responsible for ing infection with Ad-mda-7, expression of the tumor this delay. To understand further the role of caspases in suppressor gene p53, whose expression has been shown Ad-mda-7-induced cell killing, H1299 cells when infected to induce apoptosis,12 increased in p53wt, A549 and H460 with Ad-mda-7 in the presence of pan-caspase inhibitor tumor cells, but was undetectable in p53null H1299 cells. Z-VAD.FMK, a broad spectrum cell-permeable caspase Although p53 expression seemed slightly up-regulated in inhibitor, resulted in inhibition of apoptosis. This obser- Ad-Luc-infected p53wt cells (A549 and H460) as com- vation suggests caspases are required for inducing pared with PBS control, a 5.4-fold increase in p53 apoptosis mediated by mda-7. The inability to achieve expression was observed in Ad-mda-7-infected tumor complete inhibition of apoptosis might be due either to cells when compared with Ad-Luc-infected cells. These degradation of the inhibitor or to the presence of other results suggest p53 up-regulation by Ad-mda-7 is specific caspases that are yet to be identified. Meanwhile, the key and not due to nonspecific viral infection.13 While the molecules underlying the selection of apoptotic pathways molecular mechanism responsible for up-regulation of versus cell cycle arrest, the mechanism of cell cycle arrest, p53 is not well understood, it is believed that most of it and the mechanism of the p53-independent apoptotic is due to post-transcriptional regulation via stabilization pathway still remain unresolved. Furthermore, the differ- of p53 protein.14 ences in response against the apoptotic signal between In light of the recent demonstration of Bax up- tumor cells and normal cells are not well known. How- regulation in Ad-mda-7-infected breast cancer cell lines ever, these results imply there are differences in the TD47 and MCF-7,5 we examined our lung tumor cell lines mechanisms to elicit the apoptotic pathway. At least, of choice for Bax and Bak expression and observed a mda-7 expression mediated by adenovirus does not trig- modest up-regulation of Bax and Bak following Ad-mda- ger keys to activate the apoptotic pathway in NHLF cells. 7 infection in p53 wild-type lung cancer cell lines (A549 In the clinical setting, tumors may carry a diverse array and H460). This observation is consistent with the p53- of mutated tumor suppressor genes, oncogenic genes, or dependent pathway of apoptosis, wherein Bax is directly both. Therefore, our finding that Ad-mda-7 was able to transcriptionally induced by p53, resulting in the release induce apoptosis in a series of lung cancer cell lines that of cytochrome c from the mitochondria and activation of utilize a variety of mutated apoptotic signaling pathways the downstream caspase cascade.14,15 Together, these has clear clinical implications. Because of its broad spec- results suggest that up-regulation of Bax and Bak protein trum of activity, Ad-mda-7 is a likely prototype for a following mda-7 expression is dependent on a functional novel class of tumor suppressor genes with the potential wild-type p53 gene and that the mda-7 and p53 pathways to become new therapeutic agents for clinical cancer gene may converge on common downstream apoptotic effec- therapy and the potential to extend the repertoire of anti- tors. This hypothesis, in turn, is supported by a report tumor gene therapy agents for treating lung cancer. that Ad-p53-mediated wild-type p53 transfer up-regu- lated Bax and Bak protein expression in p53-mutated and Materials and methods p53-deficient lung cancer cell lines.16 However, since we observed no increase in the levels of Bax and Bak protein Cell culture in p53-deficient H1299 cells undergoing apoptosis, we Human non-small cell lung carcinoma (NSCLC) cell lines concluded that mda-7-mediated tumor cell killing occurs A549 (adenocarcinoma) and H1299 (large cell carcinoma)

Gene Therapy Ad-mda-7 in lung cancer tumor suppression T Saeki et al 2056 were gifts from Dr A Gazdar and Dr JD Minna treated with PBS. Forty-eight hours after infection, cells (University of Texas South Western Medical Center, were harvested by trypsinization, fixed with 70% ethanol, Dallas, Texas), H460 (large cell carcinoma) was obtained and resuspended in 500 ␮l of a propidium iodide (PI) from the American Type Culture Collection (Bethesda, solution (Boehringer Mannheim, Indianapolis, IN, USA) MD, USA). The p53 status for A549 and H460 is wild (5 ␮g/ml PI and 10 ␮g/ml RNase). DNA contents and type (p53wt) and for H1299 is null (p53null). All cancer cell cycle phases were analyzed using a fluorescence-acti- cells were maintained in RPMI 1640 medium containing vated cell sorter (EPICS XL-MCL; Beckman Coulter, Ful- 10% fetal bovine serum, antibiotics and l-glutamine. Nor- lerton, CA, USA). mal human lung fibroblast (NHLF) cells were obtained from Clonetics (Walkersville, MD, USA) and maintained Annexin V assay according to the manufacturer’s instructions. All cells Annexin V staining was performed as described.10 were verified to be free of mycoplasma before the start Briefly, cells were seeded in 60-mm culture dishes (5 × of the experiments and were used in the log phase of 105 cells per dish), treated with PBS, Ad-mda-7 or Ad- growth. Luc and harvested by trypsinization 48 h after infection. Cells were washed with PBS and stained with annexin Construction of recombinant adenoviral vector V (PharMingen, San Diego, CA, USA) according to the Replication-deficient human type 5 adenoviral (Ad5) vec- manufacturer’s instructions and percentages of annexin tors carrying either the mda-7 gene, or a luciferase gene V-positive cells were analyzed by flow cytometry. linked to an internal CMV-IE promoter and followed by SV40 polyadenylation (pA) signal were constructed (they are referred to herein as Ad-mda-7 and Ad-Luc, TUNEL staining respectively).4 Viruses were propagated in 293 cells and Cells were seeded in two-well chamber slides at a density × 5 purified by chromatography. of 1 10 cells per well and infected with Ad-mda-7 or Ad-Luc. Seventy-two hours after infection, cells were Gene transfer analyzed for apoptosis by terminal deoxynucleotidyl Cells were plated 1 day before being infected. Tumor transferase-mediated biotinylated UTP nick-end labeling cells (A549, H460 and H1299) were infected with adeno- (TUNEL) staining with terminal transferase (Boehringer virus vectors (Ad-mda-7 or Ad-Luc) at a multiplicity of Mannheim). infection (MOI) of 2500 viral particles per cell. NHLF cells were infected at a MOI of 2000 viral particles per cell. Western blot analysis MOI values were determined by the results of immuno- To determine the regulation of various proapoptotic histochemical staining to adjust 50–80% of cells genes (p53, Bax and Bak) and activation of caspases fol- expressing mda-7 protein. lowing mda-7 expression, Western blot analysis was per- formed as described previously.19 Briefly, cells were har- Immunohistochemical staining vested by trypsinization and resuspended in lysis buffer Immunohistochemical staining was carried out on virus- (62.5 mm Tris-HCl, 2% SDS, 10% glycerol, 4 m urea). Pro- infected cells to determine mda-7 protein expression. tein samples (50 ␮g) were each diluted into a 20 ␮l sol- Briefly, cells were seeded in two-well chamber slides at ution of lysis buffer and 5% 2-mercaptoethanol (Bio-Rad × 5 a density of 1 10 cells per well and infected with Ad- Laboratories, Hercules, CA, USA) and heated in a water mda-7 or Ad-Luc. Forty-eight hours after infection, cells bath at 95°C for 5 min. Then, protein extracts were separ- were fixed in a 4% formalin solution. After blocking of ated by 10% SDS-PAGE in a vertical-slab gel electro- endogenous peroxidase activity with 0.3% H2O2 in meth- phoresis cell (Bio-Rad). Next, the separated proteins were anol, cells were incubated with normal goat serum. Fol- transferred from gel to nitrocellulose membrane lowing incubation, slides were treated with rabbit poly- (Hybond-ECL; Amersham Pharmacia Biotech, Bucking- clonal anti-mda-7 antibody (1:5000 dilution). After hamshire, UK) and then blocked in a blocking solution incubation with an anti-rabbit secondary antibody (ABC (5% dry milk and 0.3% Tween 20 in PBS) for 1 h. Then, kit, Vector Laboratories, Burlingame, CA, USA), membranes were incubated with the primary expression of mda-7 in cells was detected with DAB p53 (1:1000), Bax (1:500), Bak (1:500), Bcl-2 (1:300), cas- (Sigma, St Louis, MO, USA) by enhancement with an pase-9 (1:2000), caspase-3 (1:1000), PARP (1:250), and ␤- avidin–biotin reaction ABC kit (Vector Laboratories). actin (1:10000). (p53, Bax, Bak and Bcl-2 antibodies are from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Determination of cell growth rate caspase-9, caspase-3, and PARP antibodies from Phar- Cancer and normal cell lines used in this study were Mingen; and ␤-actin antibody from Sigma.) The mem- plated in six-well tissue culture dishes at a density of 5 × 4 branes were then incubated with horseradish peroxidase- 10 cells per well. Tumor cells and NHLF cells were labeled secondary antibodies (Amersham). Finally, the then infected with Ad-mda-7 or Ad-Luc, or treated with proteins were visualized on enhanced chemilumi- PBS (as a negative control). Cells in each treatment group nescence film (Hyperfilm; Amersham) by application of were plated in triplicate and cultured for 5 days. Then, Amersham’s Enhanced Chemiluminescence Western at designated time-points, cells were harvested by tryp- Blotting Detection System. sinization, and stained with trypan blue (GIBCO BRL, Grand Island, NY, USA), to reveal dead cells. Viable cells were then counted on a hemocytometer. Acknowledgements Cell cycle analysis We thank Peggy James for the preparation of the manu- Cells were seeded in 10-cm culture dishes (1 × 105 cells script. This work was supported in part by a Specialized per dish) and infected with Ad-mda-7 or Ad-Luc, or Program of Research Excellence (SPORE) in Lung Cancer

Gene Therapy Ad-mda-7 in lung cancer tumor suppression T Saeki et al 2057 (P50-CA70907) (JA Roth) and by a sponsored research 9 Harris MP et al. Adenovirus-mediated p53 gene transfer inhibits agreement with Introgen Therapeutics, Inc. growth of human tumor cells expressing mutant p53 protein. Cancer Gene Ther 1996; 3: 121–130. 10 Perkins CL et al. The role of Apaf-1, Caspase-9, and Bid proteins References in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. Cancer Res 2000; 60: 1645–1653. 1 Jiang H et al. Subtraction hybridization identifies a novel mela- 11 Sun XM et al. Distinct caspase cascades are initiated in receptor- noma differentiation associated gene, mda-7, modulated during mediated and chemical-induced apoptosis. J Biol Chem 1999; 274: human melanoma differentiation, growth and progression. 5053–5060. 1995; 11: 2477–2486. 12 Dragovich T, Rudin CM, Thompson CB. 2 Jiang H et al. The melanoma differentiation-associated gene pathways that regulate cell survival and cell death. Oncogene mda-6, which encodes the cyclin-dependent kinase inhibitor 1998; 17: 3207–3213. p21, is differentially expressed during growth, differentiation 13 McPake CR et al. Wild-type p53 induction mediated by repli- and progression in human melanoma cells. Oncogene 1995; 10: cation-deficient adnoviral vectors. Cancer Res 1999; 59: 4247– 1855–1864. 4251. 3 Lin JJ, Jiang H, Fisher PB. Melanoma differentiation associated 14 Miyashita T, Reed JC. Tumor suppressor p53 is a direct tran- gene-9, mda-9, is a human gamma interferon responsive gene. scriptional activator of the human Bax gene. Cell 1995; 80: Gene 1998; 30: 105–110. 293–299. 4 Jiang H et al. The melanoma differentiation associated gene mda- 15 Cregan SP et al. Bax-dependent caspase-3 activation is a key 7 suppresses cancer cell growth. Proc Natl Acad Sci USA 1996; determinant in p53-induced apoptosis in . J Neurosci 93: 9160–9165. 1999; 19: 7860–7869. 5SuZZet al. The cancer growth suppressor gene mda-7 selectively 16 Pearson AS et al. Up-regulation of the proapoptotic mediators induces apoptosis in human breast cancer cells and inhibits Bax and Bak after adenovirus-mediated p53 gene transfer in tumor growth in nude mice. Proc Natl Acad Sci USA 1998; 95: lung cancer cells. Clin Cancer Res 2000; 6: 887–890. 14400–14405. 17 Rampino N et al. Somatic frameshift mutations in the BAX gene 6 Adams JM, Cory S. The bcl-2 protein family: arbiters of cell sur- in colon cancers of the microsatellite mutator phenotype. Science vival. Science 1998; 281: 1322–1326. 1997; 275: 967–969. 7 Swisher SG et al. Adenovirus-mediated p53 gene transfer in 18 Zou H, Li Y, Liu X, Wang X. An APAF-1–cytochrome c multi- advanced non-small-cell lung cancer. J Natl Cancer Inst 1999; 91: meric complex is a functional apoptosome that activates pro- 763–771. caspase-9. J Biol Chem 1999; 274: 11549–11556. 8 Cleyman GL, Frank DK, Bruso PA, Goepfert H. Adenovirus- 19 Ji L et al. Induction of apoptosis and inhibition of tumorigenicity mediated wild-type p53 gene transfer as a surgical adjuvant in and tumor growth by adenovirus vector-mediated fragile histi- advanced head and neck cancers. Clin Cancer Res 1999; 5: dine triad (FHIT) gene overexpression. Cancer Res 1999; 59: 1715–1722. 3333–3339.

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