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Adenovirus-Mediated mda-7 Gene Expression Radiosensitizes Non-Small Cell Lung Cancer Cells via TP53-Independent Mechanisms
doi:10.1006/mthe.2002.0714, available online at http://www.idealibrary.com on IDEAL
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Adenovirus-Mediated mda-7 Gene Expression Radiosensitizes Non-Small Cell Lung Cancer Cells via TP53-Independent Mechanisms Shinichiro Kawabe,1 Takashi Nishikawa,1 Anupama Munshi,1 Jack A. Roth,2 Sunil Chada,3 and Raymond E. Meyn1,* Departments of 1Experimental Radiation Oncology and 2Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030, USA 3 Introgen Therapeutics Inc., 2250 Holcombe Blvd., Houston, Texas 77030, USA *To whom correspondence and reprint requests should be addressed. Fax: (713) 794-5369. E-mail: [email protected].
We examined the ability of adenoviral-mediated expression of the melanoma differentiation associated gene-7 (Ad-mda-7), to radiosensitize non-small cell lung cancer (NSCLC) cell lines (A549 (wt-TP53/wt-RB1) and H1299 (del-TP53/wt-RB1)), and normal human lung fibroblast (NHLF) lines (CCD-16 and MRC-9). Results of clonogenic assays indicated that Ad-mda7 enhanced the radiosensitivity of the NSCLC cells independent of their TP53 gene status. On the other hand, the NHLF cell lines seemed to be relatively resistant to the cytotoxic effects of Ad-mda7 and were not radiosensitized compared with the NSCLC cells. We further examined the basis for this difference in the ability of Ad-mda7 to radiosensitize NSCLC cells compared with normal cells. Radiation-induced apoptosis was restored in the NSCLC lines, but not in the normal lines. Western blot analysis revealed that Ad-mda7 enhances radiosensitivity independently of any ability to upregulate the expression of Fas or Bax in NSCLC cells. Further analysis indicated that phosphorylated c-Jun expression was increased by Ad-mda7 in both A549 and H1299 cells, but not in CCD-16 cells. These results support the use of gene replacement with Ad-mda7 in combination with radiotherapy for the treatment of NSCLC. Key Words: apoptosis, adenovirus, mda-7, radiation, lung cancer, c-Jun
INTRODUCTION The lack of cell differentiation resulting in uncontrolled cell growth and reduced apoptosis propensity may contribute to the neoplastic transformation of cells [1,2]. Therefore, induction of cell differentiation may represent an alternative or adjuvant to conventional cancer therapies. For example, several melanoma differentiation-associated (mda) genes have been isolated that may influence human melanoma cell growth and differentiation [3,4]. Melanoma differentiation associated gene-7, mda-7, was identified by virtue of its enhanced expression in human melanoma cells treated with interferon (IFN-) plus the antileukemic compound mezerein [5]. The further demonstration that mda-7 mRNA is expressed in normal melanocytes and early stages of melanoma, but lost during melanoma progression, has suggested that mda-7 may be a novel tumor-suppressor gene [5]. The mda-7 gene encodes a novel protein of 206 amino acids with a predicted molecular weight of 23.8 kDa [5]. The protein sequence of MDA-7 indicates no significant homology to
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any previously identified protein and does not contain sequence motifs that might suggest its mechanism of action. However, a region of the MDA-7 protein displays homology to human interleukin-10 (IL-10), suggesting that MDA-7 may be a cytokine [5]. The mda-7 gene has been mapped to 1q32, where it is located within a “cytokine cluster” encompassing several IL10-related genes [6]. This cluster includes IL10, IL19, IL20, and mda7 [7]. Recently, mda-7 has been classified as IL24 [8] Studies by Fisher and his colleagues have shown that enforced expression of mda-7 suppressed cancer cell growth and induced apoptosis in human breast cancer cells [9]. Jiang et al. report that mda-7 is a potent growth suppressor gene in cells derived from a variety of tumor types including melanomas, gliomas, and cancers of the breast, colon, cervix, and prostate [10]. However, these investigations also demonstrated that expression of mda7 in normal cells (human mammary epithelial cells) has limited cytotoxic effects. Additional work from this group showed that ex vivo treatment using adenoviral-mediated
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FIG. 1. Radiosensitization by Ad-mda7 determined on the basis of clonogenic survival assays. Vector concentrations used for Ad-mda7 and Ad-Luc were 1000 vp/cell for the A549 cell line (A), 250 for the H1299 cell line (B), and 1500 for the CCD16 (C) and MRC-9 (D) cell lines. Radiation was given 48 hours after transfection. Each data point represents the average of three independent experiments. Symbols represent mock infection (filled diamond), Admda7 (filled square), and Ad-Luc (filled triangle). Bar: SE
doi:10.1006/mthe.2002.0714, available online at http://www.idealibrary.com on IDEAL
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mda-7 (Ad-mda7) inhibited the growth of MCF-7 xenograft tumors in nude mouse models. The resistance of tumor cells to the cytotoxic effects of ionizing radiation remains the key limitation to the use of radiotherapy for the treatment of human cancer. To overcome such resistance, we have recently examined gene therapy strategies that radiosensitize non-small cell lung cancer (NSCLC) cells by restoring apoptosis. Two vectors, Ad-p53 and Ad-p16, have shown promise in this regard [11,12]. However, in our search for better radiosensitizing vectors, we have turned our attention to Ad-mda7 because of its documented ability to restore apoptosis propensity in NSCLC cells [13]. The purpose of the present study was to determine whether Ad-mda7 enhances the radiosensitivity of NSCLC cells. We have also examined the apoptosis pathway mediated by the combination of Ad-mda7 and irradiation.
RESULTS Ad-mda7 Enhances Radiosensitivity of NSCLC Cells, but Not NHLF Lines We first tested whether Ad-mda7 infection sensitizes NSCLC cells to irradiation in vitro. Clonogenic assays were performed on two NSCLC lines, A549 and H1299, and two normal human lung fibroblast (NHLF) cell lines, CCD16 and MRC-9. These lines were infected with either Admda7 or Ad-Luc (control vector) and irradiated 48 hours later. The 48-hour time course was based on cell cycle
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analysis that demonstrated maximum G2 arrest in this time frame. Ad-mda7 radiosensitized both NSCLC cell lines even at the clinically relevant dose of 2 Gy (Fig. 1). For example, the percent survival for A549 cells at 2 Gy was reduced from 69.8 ± 3.1% to 38.5 ± 3.2% (Fig. 1A) and a dose reduction factor (DRF) calculated at the 50% survival level for Ad-mda7 plus radiation in A549 cells was 1.93. The percent survival for H1299 cells at 2 Gy was reduced from 78.2 ± 3.7% to 45.7 ± 4.5% (Fig. 1B) and the DRF for H1299 cells was 2.06. The control vector, Ad-Luc, had no sensitizing effect for either A549 or H1299 cells when used at identical vector concentrations. On the other hand, Admda7 did not radiosensitize the NHLF lines at the clinically relevant dose of 2 Gy. The percent survival for CCD16 cells treated with radiation alone and radiation plus Ad-mda7 at 2 Gy was 43.6 ± 7.0% and 45.4 ± 3.4%, respectively (Fig. 1C), and for MRC-9 cells was 24.2 ± 3.4% and 27.2 ± 1.6%, respectively (Fig. 1D). Ad-mda7 Induces Apoptosis in NSCLC Cells, but Not Normal Cells We used the TUNEL assay to measure the level of apoptosis (Fig. 2). The percentages of TUNEL-positive cells in A549 cells (Fig. 2A), H1299 cells (Fig. 2B), CCD-16 cells (Fig. 2C), and MRC-9 cells (Fig. 2D) treated with either mock infection, 5 Gy alone, Ad-Luc alone, Ad-Luc plus 5 Gy, Ad-mda7 alone, or Ad-mda7 plus 5 Gy are shown in Fig. 2. Radiation alone resulted in an increase to 11% in the proportion of TUNEL-positive cells compared with
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control in the A549 cells. This effect was less apparent in the H1299 cells. As expected, Ad-mda7 infection alone modestly increased the proportion of TUNEL-labeled cells to 10% in A549 cells and 18% in the H1299 cells. However, the combination of Ad-mda7 and radiation produced a greater-than-additive increase in TUNEL-positive cells in both NSCLC lines, achieving levels of 38% and 35% in A549 and H1299 cells, respectively. This enhancement of radiation-induced apoptosis was not evident A when Ad-mda7 was replaced with Ad-Luc. On the other hand, TUNEL-positive cells for CCD16 (Fig. 2C) and MRC-9 (Fig. 2D) treated with Ad-mda7 alone were not substantially increased compared with controls and the combination treatment, Ad-mda7 plus 5 Gy, only slightly increased the proportion of TUNEL-positive cells in the NHLF lines. B Ad-mda7 Arrests Cells in the G2/M Phase of the Cell Cycle A previous report indicated that Ad-mda7 suppresses proliferation and induces a G2/M cell cycle arrest in NSCLC cell lines [13]. We validated these effects and examined the expression of two proteins known to be involved in cell cycle regulation, pRb and cyclin B1. Western blot analysis demonstrated that MDA-7 protein began to be expressed in both A549 and H1299 cell lines by 24 hours after
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FIG. 2. Apoptosis assessed by TUNEL assay for A549 (A), H1299 (B), CCD-16 (C), and MRC-9 (D) cells. Cells were irradiated 48 hours following transfection and harvested 2 days after irradiation or 4 days after transfection. Vector concentrations used were identical to those used for Fig. 1. Each data point represents the average of two independent experiments. Bar: SE.
infection (Fig. 3A). Multiple bands were evident due to the detection of glycosylated forms of the protein [8,14]. As MDA-7 protein began to be expressed following Ad-mda7 administration, expression of pRb declined within 2–4 days in A549 and H1299 cells (Fig. 3B). In contrast, cyclin B1 expression was slightly upregulated by day 2, but these levels declined to levels below control by day 3 in A549 or day 4 in H1299 cells. These results suggested that the
FIG. 3. Western blot analysis of MDA-7 (A), cyclin B1, and pRb (B) protein expression levels as a function of time in A549 and H1299 cells infected with Admda7. Cells were harvested at different times after infection. Vector concentrations used were identical to those used for Fig. 1. Blots shown are representative of at least two independent experiments. Actin was used as a loading control.
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cells may accumulate in G2/M phase approximately 2 days following Ad-mda7 transfection. This was confirmed by flow cytometry analysis of A549 and H1299 cells at 2 days following Ad-mda7 treatment (Fig. 4A). We then tested whether G2/M arrest by itself has a role in enhancing the radiosensitivity of these cells. Nocodazole, a drug that reversibly blocks microtubule polymerization, was used to accumulate A549 and H1299 cells in G2/M. The treatment schedule for Nocodazole (200 ng/ml) to induce the same degree of G2/M arrest compared with Ad-mda7 was 4 hours for A549 cells and 3.5 hours for H1299 cells. We then determined the radiosensitivity of A549 and H1299 cells treated with Nocodazole compared with controls, using clonogenic assays. The results indicate that G2/M arrest by itself, at least to the degree mediated by Ad-mda7, does not enhance the radiosensitivity of NSCLC cells (Fig. 4B). Ad-mda7 Enhances Radiosensitivity Independent of p53, Bax, and Fas We analyzed the expression of p53, Bax, and Fas protein in A549 and H1299 following treatment with either radiation alone, Ad-mda7 alone, Ad-mda7 plus radiation, AdLuc, or Ad-Luc plus radiation (Fig. 5A). The p53 protein levels dramatically increased in A549 cells treated with either radiation or Ad-mda7, but not with Ad-Luc. Fas protein expression in A549 cells treated with either radiation or Ad-mda7 was also increased and this effect was consistent
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FIG. 4. Ad-mda7 arrests cells in G2/M. (A) Cell cycle analysis of A549 and H1299 treated with either Admda7 or Nocodazole (200 ng/ml). The dose and exposure time of Nocodazole to accumulate the same proportion of cells in G2/M phase as was present 48 hours after Ad-mda7 transfection was determined in preliminary experiments. Data shown are representative of two independent experiments. (B) Clonogenic survival assays to determine radiosensitization by G2/M arrest induced by Nocodazole (200 ng/ml). Radiation was given after 4 hours of Nocodazole exposure for the A549 cell line, and after 3.5 hours of Nocodazole exposure for the H1299 cell line. Symbols represent radiation alone (filled diamond) and Nocodazole (open square). Bar: SE.
with a dependence on wild-type p53, as Fas protein expression in H1299 cells (which do not express p53) was not enhanced by these treatments. On the other hand, Bax protein expression was not significantly changed in either cell line by these treatments. Thus, because A549 and H1299 cells are equally radiosensitized by Ad-mda7, neither p53, Fas, or Bax correlated with radiosensitization by Ad-mda7. Ad-mda7 Enhances the Expression of p-c-Jun Protein It has been reported that radiation-induced apoptosis requires the activation of c-Jun N-terminal kinase (JNK) [15,16]. We tested whether Ad-mda7 was able to activate JNK and whether this correlated with radiosensitization. Rb, p-c-Jun, and JNK-1 protein levels were determined in A549, H1299, and CCD-16 cell lines treated with radiation alone, Ad-mda7 alone, Ad-mda7 plus radiation, Ad-Luc, or Ad-Luc plus radiation (Fig. 5B). Rb protein expression was reduced in A549 and H1299 cells treated with Ad-mda7, but was not changed with control vector in these cell lines. The expression of both p-c-Jun and JNK-1 was enhanced in A549 and H1299 cells treated with Ad-mda7. In CCD16 cells, however, Rb protein expression was slightly reduced, but expression of p-c-Jun and JNK-1 was not enhanced by Ad-mda7 treatment. These results are consistent with the possibility that Ad-mda7 mediates radiosensitivity and enhances apoptosis through the activation of JNK-1 and the subsequent activation of p-c-Jun.
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FIG. 5. Ad-mda7 enhances the expression of p-c-Jun but not Bax. (A) Western blot analysis of p53, Fas, and Bax protein expression in A549 and H1299 cells treated with either 5 Gy alone, Ad-mda7 alone, Ad-mda7 plus 5 Gy, Ad-Luc alone, or Ad-Luc plus 5 Gy. Cells were harvested 1 day after irradiation or 3 days after transfection. Vector concentrations used were identical as those used for Fig. 1. Actin was used as a loading control. (B) Western blot analysis of Rb, phosphorylated c-Jun, and MDA-7 protein in A549, H1299, and CCD16 cell lines treated with 5 Gy alone, Ad-mda7 alone, Ad-mda7 plus 5 Gy, AdLuc alone, or Ad-Luc plus 5 Gy. Cells were harvested 1 day after irradiation or 3 days after transfection. Vector concentrations used were identical as those used for Fig. 1. Actin was used as a loading control.
Curcumin Abrogates Ad-mda7 Mediated Radiosensitization Curcumin, a dietary pigment responsible for the yellow color of curry, has been reported to inhibit JNK activation [17]. We therefore determined the expression of p-c-Jun protein in A549 and H1299 cells treated with radiation alone, curcumin alone, Ad-mda7 alone, radiation plus curcumin, radiation plus Ad-mda7, or radiation plus curcumin plus Ad-mda7 (Fig. 6A). Curcumin when used alone enhanced p-c-Jun expression, as did Ad-mda7 used alone. However, curcumin reduced Ad-mda7 mediated activation of p-c-Jun in irradiated and unirradiated cells. To examine whether curcumin inhibits Ad-mda7 mediated radiosensitivity, we carried out clonogenic assays using A549 and H1299 lines. Cells were infected with Ad-mda7 and irradiated 48 hours later. Curcumin abrogated Ad-mda7 radiosensitization in both cell lines (Fig. 6B).
DISCUSSION We have compared the abilities of Ad-mda7 to radiosensitize NSCLC cell lines that have either wt-TP53 or deleted TP53 status and NHLF cell lines. The clonogenic survival results indicated that both the A549, which have wt-TP53,
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and the H1299, which is deleted for wt-TP53, cell lines were substantially radiosensitized by Ad-mda7 (Figs. 1A and 1B). However, two different NHLF cell lines were not (Figs. 1C and 1D). These results were consistent with the analysis of apoptosis by TUNEL (Fig. 2), suggesting that the radiosensitizing effect of Ad-mda7 in NSCLC cells involves a restoration of apoptosis propensity. Moreover, as both A549 and H1299 cells were equally affected, this activity of Ad-mda7 seems to be independent of the endogenous TP53 status of the cells. We also investigated whether this radiosensitizing effect of Ad-mda7 was due to an alteration of cell cycle phase distribution. Tumor cells are typically sensitive to radiation killing in the G2/M phase of the replicative cell cycle [18]. The transition from G2 to M is regulated in part by the mitosis-promoting factor, consisting of cyclin B1 and a G2-specific cyclin-dependent kinase Cdc2 [19]. Normally, cyclin B1 levels gradually increase as cells progress into the S and G2 phases of the cell cycle. As MDA-7 protein is expressed in these cell lines, the expression of cyclin B1 increases, peaking 2 days following Ad-mda7 transfection (Fig. 3), and the cells accumulate in G2/M (Fig. 4). Thus, there was the possibility that the radiosensitivity of the NSCLC lines might be enhanced solely by this accumulation in G2/M. We
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FIG. 6. Curcumin abrogates Ad-mda7 mediated radiosensitization. (A) Western blot analysis of p-c-Jun in A549 and H1299 cells treated with 5 Gy alone, curcumin (10 M), curcumin plus 5 Gy, Ad-mda7, Ad-mda7 plus 5 Gy, curcumin plus Ad-mda7, or curcumin plus Ad-mda7 plus 5 Gy. Cells were harvested 1 day after irradiation or 3 days after transfection. Curcumin was added 1 day after transfection. Actin was used as a loading control. (B) Clonogenic survival assays to determine radiosensitization in A549 and H1299 cells treated with either curcumin or curcumin plus Ad-mda7. Radiation was given 2 days after transfection. Curcumin was added 1 day after transfection. The vector concentrations used were identical to that used for Fig. 1. Each data point represents the average of three independent experiments. Bar: SE.
examined whether this degree of accumulation of G2/M phase has an important role in enhancing the radiosensitivity of these NSCLC lines by mimicking this effect with Nocodazole. Accumulating cells in G2/M with a dose of Nocodazole to a degree equal to or above that achieved with Ad-mda7 did not produce a radiosensitizing effect (Fig. 4B). These results indicate that the G2/M arrest produced by Ad-mda7 is not the principal cause of its radiosensitizing effect. We then investigated the expression of other proteins involved in apoptosis by western blot analysis. The expression of the pro-apoptotic protein Bax was not upregulated in either the A549 or H1299 cell lines, although the expression of p53 and Fas protein was increased in the A549 line, but not in H1299 (Fig. 5A). Thus, the changes in expression of these proteins do not correlate with the radiosensitizing effects of Ad-mda7. Other investigators have reported increased Bax protein after Ad-mda7 treatment of lung and breast cancer cells [9,13]. However, the dose of Ad-mda7 used in the present study was lower than previously described. In addition, Ad-mda7 has activity against Bax-deficient DU-145 cells, further suggesting that Bax upregulation is not a prerequisite for the effects of Admda7 [20].
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Rb is a negative regulator of apoptosis and suppresses the JNK/SAPK signaling pathway. This inhibition of JNK/SAPK activity may be an important mechanism by which Rb negatively regulates the stress-activated cellular events leading to cell death [21]. Rb has been shown to suppress radiationinduced apoptosis and, when Rb’s function is antagonized, apoptosis can proceed [22]. It is well established in the literature that activation of JNK is an important step in cells undergoing apoptosis in response to radiation [15,16]. Expression of Rb was suppressed in both A549 and H1299 cell lines following treatment with Ad-mda7, whereas JNK and c-Jun were concordantly activated (Fig. 5B). Based on these findings, it is possible that the activation of c-Jun and JNK-1 is mediated by the reduction of Rb protein expression induced by Ad-mda7. However, we also observed that JNK activation does not take place in the NHLF lines in spite of a reduction in Rb expression. Jiang et al. [10] have reported that Ad-mda7 suppresses the growth of cancer cell lines with defects in the Rb gene. Thus, activation of JNK and pc-Jun, but not suppression of Rb, correlates with the radiosensitizing effect of Ad-mda7. A role for activation of JNK in the radiosensitizing effect of Ad-mda7 was tested using curcumin, a non-specific inhibitor of JNK activation [17]. Curcumin suppressed the ability of Ad-mda7 to
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upregulate the expression of p-c-Jun in A549 and H1299 cells, consistent with an inhibition of JNK activity (Fig. 6A). Furthermore, neither cell line was radiosensitized when Admda7 was combined with curcumin (Fig. 6B). These data are consistent with, but do not prove, the possibility that Admda7 radiosensitizes NSCLC cells through activation of JNK and p-c-Jun. This appears to be blocked in normal cells, which are not radiosensitized by Ad-mda7. The mechanism by which JNK and p-c-Jun become activated by Ad-mda7 is not known, however, this may be due to a general activation of signal transduction pathways. Ad-mda7 has previously been shown to activate p38 [23] and PKR [24]. MDA7 protein also has been shown to activate STAT-3 [25]. In addition to two other vectors that we have previously examined, Ad-p53 and Ad-p16, Ad-mda7 has potent radiosensitizing properties. On A549 cells, Ad-mda7 seems to be a better radiosensitizer; Ad-mda7 produces a DRF of 1.93 compared with a DRF of 1.37 for Ad-p53 and a DRF of 1.75 for Ad-p16 [11,12]. This is further evidence to indicate that Ad-mda7 may radiosensitize through a novel mechanism that does not depend on p53. In light of this possibility, it would be of interest to test for an additive effect of the combination of Ad-p53 and Ad-mda7. This is currently under investigation. We have demonstrated that Ad-mda7 enhances the radiation sensitivity of NSCLC cells, but not of NHLF cells. Whether this occurs through activation of c-Jun has yet to be conclusively proven, but this possibility will be the subject of further studies. Our results indicate that the combination of Ad-mda7 and irradiation may be a useful gene therapy strategy for the treatment of NSCLC.
MATERIALS
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Cell culture, vectors, and chemicals. The human NSCLC cell lines, A549 (wt-TP53/wt-RB1) and H1299 (del-TP53/wt-RB1), and normal human lung fibroblast lines (NHLF), CCD-16 and MRC-9, were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained as specified by ATCC. The recombinant adenoviral vector (Ad-mda7) contains the CMV promoter, wild-type mda-7 cDNA, and an SV40 polyadenylation signal in a minigene cassette inserted into the E1-deleted region of modified Ad5. Adenovirus-mediated luciferase (Ad-Luc) was used as a control vector. These vectors have been described previously [6]. The preparations were tested for and determined to be free of replication-competent adenovirus and mycoplasma. Curcumin and Nocodazole were purchased from Sigma-Aldrich (Poole, UK). Stock solutions of curcumin (10 mM) were prepared freshly on the day of the experiment by dissolving the compound in ethanol. Mocktreated cells received the same concentration of ethanol. It was then diluted into medium at a concentration of 10 mM. Stock solutions of Nocodazole (5 mg/ml) were prepared by dissolving the compound in DMSO. It was then was diluted into medium (200 ng/ml). Gene delivery. In vitro transfection studies for all cell lines were performed by plating 2 ⫻ 105 cells in T25 flasks. Forty-eight hours after plating, cells were incubated for 1 hour with purified vector in 1 ml of medium without serum. After 1 hour, fresh medium supplemented with 10% FBS was added to the flask. Serum-free medium was used for mock transfection. Cells were further incubated for 48 hours before survival curves were generated.
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Radiation and clonogenic assay. Cells were irradiated with a high-dose rate 137Cs unit (3.7 Gy/minute) at room temperature in T25 flasks. Irradiation was performed 48 hours after vector treatment. The effectiveness of treatments was assessed by clonogenic assays. Briefly, monolayers of A549, H1299, CCD-16, and MRC-9 cells were treated in T25 flasks as described above, and after various doses of irradiation the cells were trypsinized and counted. Known numbers of cells were then replated in 100-mm culture dishes and returned to the incubator to allow macroscopic colony development. Colonies were counted after 10–14 days, and the percent plating efficiency and surviving fractions following given treatments were calculated based on the survival of nonirradiated cells treated with either mock infection, Ad-Luc or Ad-mda7. The vector treatments used were adjusted for each line to yield identical reductions in plating efficiency with Ad-mda7 (80%). The vector concentrations used, therefore, were 1000 vp/cell for A549, 250 vp/cell for H1299, and 1500 vp/cell for CCD-16 and MRC-9 cells. These treatments produced nearly 100% transfection efficiency. Some of the experiments on A549 cells used a different lot of Ad-mda7 vector which required 2000 vp/cell to achieve the same transfection efficiency. Apoptotic index and cell cycle analysis. Apoptosis was quantified by flow cytometry using the APO-BRDU Kit (Pharmingen, San Diego, CA). Briefly, 2 ⫻ 106 cells were fixed with 1% paraformaldehyde in PBS for 15 minutes at room temperature, washed twice with PBS, and stored in 70% ethanol at –20⬚C. For analysis, cells were incubated in the DNA labeling solution overnight at room temperature. Fluorescent labeled anti-BrdU antibody solution was added, and the cells incubated in the dark for 30 minutes at room temperature. The stained cells were analyzed by flow cytometry with the use of an EPICS flow cytometer (Coulter Corp., Hialeah, FL). All steps were performed according to the manufacturer’s recommendations. An analysis region was set based on the negative control, and the percentage of labeled cells was calculated from this region. We analyzed apoptotic indices 2 days after irradiation with 5 Gy or 4 days after infection. This time course was based on preliminary indications of the time for maximum apoptotic response. As before, infections with either Ad-mda7 or Ad-Luc were performed 48 hours before irradiation. Western analysis. Briefly, cells were scraped from the plates, washed with PBS, and lysed in cell lysis buffer. Protein (30 g) was electrophoretically separated on either 8% (for pRb), 12% (for Cyclin B1, pc-Jun, Fas, Bax and p53), or 15% (for MDA-7) SDS-polyacrylamide gels and transferred to polyvinylide difluoride membrane (Millipore, Bedford, MA). Mouse monoclonal antibodies for pRb, Cyclin B1 (Pharmingen, San Diego, CA), p53 (DAKO, Carpinteria, CA), and Fas (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal antibody for Bax (Santa Cruz Biotechnology, Santa Cruz, CA), MDA-7 (Introgen Therapeutics Inc., Houston, TX), JNK-1 (Promega, Madison, WI), were used as primary antibodies. Primary antibody for p-c-Jun, specific for cJun p39 phosphorylated on serine-63, was obtained from Santa Cruz Biotechnology. Primary antibody for JNK-1 detects the phosphorylated, active form of stress-activated protein kinase (SAPK), also known as cJun N terminal kinase, JNK. The membranes were enhanced by chemiluminescence using ECL western blot detection reagents (Amersham Corp, Arlington Heights, IL) according to the manufacturer’s instructions. Total cellular proteins applied to each lane were adjusted to equal concentration with BCA protein assay reagent (Bio-Rad Laboratories, Richmond, CA), and were confirmed with coomassie brilliant blue staining method.
ACKNOWLEDGMENTS This study was supported in part by grants R41 CA88421, P01 CA78778, P01 CA06294, and P30 CA16672 from the National Cancer Institute and a sponsored research agreement with Introgen Therapeutics, Inc. The vectors used in this research were the kind gift of Introgen Therapeutics, Inc. RECEIVED FOR PUBLICATION AUGUST 21, 2001; ACCEPTED SEPTEMBER 6, 2002.
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REFERENCES 1. Fisher, P. B., and Grant, S. (1985). Effects of interferon on differentiation of normal and tumor cells. Pharmacol. Ther. 27: 143–166. 2. Hoffman, B., and Liebermann, D. A. (1994). Molecular controls of apoptosis: differentiation/growth arrest primary response genes, proto-oncogenes, and tumor suppressor genes as positive & negative modulators. Oncogene 9: 1807–1812. 3. Jiang, H., and Fisher, P. (1993). Use of sensitive and efficient subtraction hybridization protocol for the identification of genes differentially regulated during the induction of differentiation in human melanoma cells. Mol. Cell. Different. 1: 285–299. 4. Jiang, H., Lin, J., and Fisher, P. (1994). A molecular definition of terminal differentiation in human melanoma cells. Mol. Cell. Different. 2: 221–239. 5. Jiang, H., Lin, J. J., Su, Z. Z., Goldstein, N. I., and Fisher, P. B. (1995). Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11: 2477–2486. 6. Mhashilkar, A. M., et al. (2001). Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol. Med. 7: 271–282. 7. Blumberg, H., et al. (2001). Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell 104: 9–19. 8. Wang, M., Tan, Z., Zhang, R., Kotenko, S. V., and Liang, P. (2002). Interleukin 24 (MDA7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL20R2. J. Biol. Chem. 277: 7341–7347. 9. Su, Z. Z., et al. (1998). The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc. Natl. Acad. Sci. USA 95: 14400–14405. 10. Jiang, H., et al. (1996). The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc. Natl. Acad. Sci. USA 93: 9160–9165. 11. Kawabe, S., Roth, J. A., Wilson, D. R., and Meyn, R. E. (2000). Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53dependent manner. Oncogene 19: 5359–5366. 12. Kawabe, S., et al. (2001). Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts. Int. J. Radiat. Biol. 77: 185–194.
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13. Saeki, T., et al. (2000). Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 7: 2051–2057. 14. Lebedeva, I. V., et al. (2002). The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 21: 708–718. 15. Chen, Y. R., Meyer, C. F., and Tan, T. H. (1996). Persistent activation of c-Jun N-terminal kinase 1 (JNK1) in gamma radiation-induced apoptosis. J. Biol. Chem. 271: 631–634. 16. Chen, Y. R., Wang, X., Templeton, D., Davis, R. J., and Tan, T. H. (1996). The role of cJun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. J. Biol. Chem. 271: 31929–31936. 17. Chen, Y. R., and Tan, T. H. (1998). Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway by curcumin. Oncogene 17: 173–178. 18. Sinclair, W. K., and Morton, R. A. (1966). X-ray sensitivity during the cell generation cycle of cultured Chinese hamster cells. Radiat. Res. 29: 450–474. 19. King, R. W., Jackson, P. K., and Kirschner, M. W. (1994). Mitosis in transition. Cell 79: 563–571. 20. Madireddi, M. T., Su, Z. Z., Young, C. S., Goldstein, N. I., and Fisher, P. B. (2000). Mda7, a novel melanoma differentiation associated gene with promise for cancer gene therapy. Adv. Exp. Med. Biol. 465: 239–261. 21. Shim, J., et al. (2000). Rb protein down-regulates the stress-activated signals through inhibiting c-Jun N-terminal kinase/stress-activated protein kinase. J. Biol. Chem. 275: 14107–14111. 22. Haas-Kogan, D. A., et al. (1995). Inhibition of apoptosis by the retinoblastoma gene product. EMBO J. 14: 461–472. 23. Sarkar, D., et al. (2002). mda-7 (IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc. Natl. Acad. Sci. USA 99: 10054–10059. 24. Pataer, A., et al. (2002). Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the doublestranded RNA-dependent protein kinase (PKR). Cancer Res. 62: 2239–2243. 25. Dumoutier, L., Leemans, C., Lejeune, D., Kotenko, S. V., and Renauld, J. C. (2001). Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J. Immunol. 167: 3545–3549.
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Adenovirus-Mediated mda-7 Gene Expression Radiosensitizes Non-Small Cell Lung Cancer Cells via TP53-Independent Mechanisms Shinichiro Kawabe,1 Takashi Nishikawa,1 Anupama Munshi,1 Jack A. Roth,2 Sunil Chada,3 and Raymond E. Meyn1,* Departments of 1Experimental Radiation Oncology and 2Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030, USA 3 Introgen Therapeutics Inc., 2250 Holcombe Blvd., Houston, Texas 77030, USA *To whom correspondence and reprint requests should be addressed. Fax: (713) 794-5369. E-mail: [email protected].
We examined the ability of adenoviral-mediated expression of the melanoma differentiation associated gene-7 (Ad-mda-7), to radiosensitize non-small cell lung cancer (NSCLC) cell lines (A549 (wt-TP53/wt-RB1) and H1299 (del-TP53/wt-RB1)), and normal human lung fibroblast (NHLF) lines (CCD-16 and MRC-9). Results of clonogenic assays indicated that Ad-mda7 enhanced the radiosensitivity of the NSCLC cells independent of their TP53 gene status. On the other hand, the NHLF cell lines seemed to be relatively resistant to the cytotoxic effects of Ad-mda7 and were not radiosensitized compared with the NSCLC cells. We further examined the basis for this difference in the ability of Ad-mda7 to radiosensitize NSCLC cells compared with normal cells. Radiation-induced apoptosis was restored in the NSCLC lines, but not in the normal lines. Western blot analysis revealed that Ad-mda7 enhances radiosensitivity independently of any ability to upregulate the expression of Fas or Bax in NSCLC cells. Further analysis indicated that phosphorylated c-Jun expression was increased by Ad-mda7 in both A549 and H1299 cells, but not in CCD-16 cells. These results support the use of gene replacement with Ad-mda7 in combination with radiotherapy for the treatment of NSCLC. Key Words: apoptosis, adenovirus, mda-7, radiation, lung cancer, c-Jun
INTRODUCTION The lack of cell differentiation resulting in uncontrolled cell growth and reduced apoptosis propensity may contribute to the neoplastic transformation of cells [1,2]. Therefore, induction of cell differentiation may represent an alternative or adjuvant to conventional cancer therapies. For example, several melanoma differentiation-associated (mda) genes have been isolated that may influence human melanoma cell growth and differentiation [3,4]. Melanoma differentiation associated gene-7, mda-7, was identified by virtue of its enhanced expression in human melanoma cells treated with interferon (IFN-) plus the antileukemic compound mezerein [5]. The further demonstration that mda-7 mRNA is expressed in normal melanocytes and early stages of melanoma, but lost during melanoma progression, has suggested that mda-7 may be a novel tumor-suppressor gene [5]. The mda-7 gene encodes a novel protein of 206 amino acids with a predicted molecular weight of 23.8 kDa [5]. The protein sequence of MDA-7 indicates no significant homology to
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any previously identified protein and does not contain sequence motifs that might suggest its mechanism of action. However, a region of the MDA-7 protein displays homology to human interleukin-10 (IL-10), suggesting that MDA-7 may be a cytokine [5]. The mda-7 gene has been mapped to 1q32, where it is located within a “cytokine cluster” encompassing several IL10-related genes [6]. This cluster includes IL10, IL19, IL20, and mda7 [7]. Recently, mda-7 has been classified as IL24 [8] Studies by Fisher and his colleagues have shown that enforced expression of mda-7 suppressed cancer cell growth and induced apoptosis in human breast cancer cells [9]. Jiang et al. report that mda-7 is a potent growth suppressor gene in cells derived from a variety of tumor types including melanomas, gliomas, and cancers of the breast, colon, cervix, and prostate [10]. However, these investigations also demonstrated that expression of mda7 in normal cells (human mammary epithelial cells) has limited cytotoxic effects. Additional work from this group showed that ex vivo treatment using adenoviral-mediated
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FIG. 1. Radiosensitization by Ad-mda7 determined on the basis of clonogenic survival assays. Vector concentrations used for Ad-mda7 and Ad-Luc were 1000 vp/cell for the A549 cell line (A), 250 for the H1299 cell line (B), and 1500 for the CCD16 (C) and MRC-9 (D) cell lines. Radiation was given 48 hours after transfection. Each data point represents the average of three independent experiments. Symbols represent mock infection (filled diamond), Admda7 (filled square), and Ad-Luc (filled triangle). Bar: SE
doi:10.1006/mthe.2002.0714, available online at http://www.idealibrary.com on IDEAL
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mda-7 (Ad-mda7) inhibited the growth of MCF-7 xenograft tumors in nude mouse models. The resistance of tumor cells to the cytotoxic effects of ionizing radiation remains the key limitation to the use of radiotherapy for the treatment of human cancer. To overcome such resistance, we have recently examined gene therapy strategies that radiosensitize non-small cell lung cancer (NSCLC) cells by restoring apoptosis. Two vectors, Ad-p53 and Ad-p16, have shown promise in this regard [11,12]. However, in our search for better radiosensitizing vectors, we have turned our attention to Ad-mda7 because of its documented ability to restore apoptosis propensity in NSCLC cells [13]. The purpose of the present study was to determine whether Ad-mda7 enhances the radiosensitivity of NSCLC cells. We have also examined the apoptosis pathway mediated by the combination of Ad-mda7 and irradiation.
RESULTS Ad-mda7 Enhances Radiosensitivity of NSCLC Cells, but Not NHLF Lines We first tested whether Ad-mda7 infection sensitizes NSCLC cells to irradiation in vitro. Clonogenic assays were performed on two NSCLC lines, A549 and H1299, and two normal human lung fibroblast (NHLF) cell lines, CCD16 and MRC-9. These lines were infected with either Admda7 or Ad-Luc (control vector) and irradiated 48 hours later. The 48-hour time course was based on cell cycle
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analysis that demonstrated maximum G2 arrest in this time frame. Ad-mda7 radiosensitized both NSCLC cell lines even at the clinically relevant dose of 2 Gy (Fig. 1). For example, the percent survival for A549 cells at 2 Gy was reduced from 69.8 ± 3.1% to 38.5 ± 3.2% (Fig. 1A) and a dose reduction factor (DRF) calculated at the 50% survival level for Ad-mda7 plus radiation in A549 cells was 1.93. The percent survival for H1299 cells at 2 Gy was reduced from 78.2 ± 3.7% to 45.7 ± 4.5% (Fig. 1B) and the DRF for H1299 cells was 2.06. The control vector, Ad-Luc, had no sensitizing effect for either A549 or H1299 cells when used at identical vector concentrations. On the other hand, Admda7 did not radiosensitize the NHLF lines at the clinically relevant dose of 2 Gy. The percent survival for CCD16 cells treated with radiation alone and radiation plus Ad-mda7 at 2 Gy was 43.6 ± 7.0% and 45.4 ± 3.4%, respectively (Fig. 1C), and for MRC-9 cells was 24.2 ± 3.4% and 27.2 ± 1.6%, respectively (Fig. 1D). Ad-mda7 Induces Apoptosis in NSCLC Cells, but Not Normal Cells We used the TUNEL assay to measure the level of apoptosis (Fig. 2). The percentages of TUNEL-positive cells in A549 cells (Fig. 2A), H1299 cells (Fig. 2B), CCD-16 cells (Fig. 2C), and MRC-9 cells (Fig. 2D) treated with either mock infection, 5 Gy alone, Ad-Luc alone, Ad-Luc plus 5 Gy, Ad-mda7 alone, or Ad-mda7 plus 5 Gy are shown in Fig. 2. Radiation alone resulted in an increase to 11% in the proportion of TUNEL-positive cells compared with
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control in the A549 cells. This effect was less apparent in the H1299 cells. As expected, Ad-mda7 infection alone modestly increased the proportion of TUNEL-labeled cells to 10% in A549 cells and 18% in the H1299 cells. However, the combination of Ad-mda7 and radiation produced a greater-than-additive increase in TUNEL-positive cells in both NSCLC lines, achieving levels of 38% and 35% in A549 and H1299 cells, respectively. This enhancement of radiation-induced apoptosis was not evident A when Ad-mda7 was replaced with Ad-Luc. On the other hand, TUNEL-positive cells for CCD16 (Fig. 2C) and MRC-9 (Fig. 2D) treated with Ad-mda7 alone were not substantially increased compared with controls and the combination treatment, Ad-mda7 plus 5 Gy, only slightly increased the proportion of TUNEL-positive cells in the NHLF lines. B Ad-mda7 Arrests Cells in the G2/M Phase of the Cell Cycle A previous report indicated that Ad-mda7 suppresses proliferation and induces a G2/M cell cycle arrest in NSCLC cell lines [13]. We validated these effects and examined the expression of two proteins known to be involved in cell cycle regulation, pRb and cyclin B1. Western blot analysis demonstrated that MDA-7 protein began to be expressed in both A549 and H1299 cell lines by 24 hours after
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FIG. 2. Apoptosis assessed by TUNEL assay for A549 (A), H1299 (B), CCD-16 (C), and MRC-9 (D) cells. Cells were irradiated 48 hours following transfection and harvested 2 days after irradiation or 4 days after transfection. Vector concentrations used were identical to those used for Fig. 1. Each data point represents the average of two independent experiments. Bar: SE.
infection (Fig. 3A). Multiple bands were evident due to the detection of glycosylated forms of the protein [8,14]. As MDA-7 protein began to be expressed following Ad-mda7 administration, expression of pRb declined within 2–4 days in A549 and H1299 cells (Fig. 3B). In contrast, cyclin B1 expression was slightly upregulated by day 2, but these levels declined to levels below control by day 3 in A549 or day 4 in H1299 cells. These results suggested that the
FIG. 3. Western blot analysis of MDA-7 (A), cyclin B1, and pRb (B) protein expression levels as a function of time in A549 and H1299 cells infected with Admda7. Cells were harvested at different times after infection. Vector concentrations used were identical to those used for Fig. 1. Blots shown are representative of at least two independent experiments. Actin was used as a loading control.
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cells may accumulate in G2/M phase approximately 2 days following Ad-mda7 transfection. This was confirmed by flow cytometry analysis of A549 and H1299 cells at 2 days following Ad-mda7 treatment (Fig. 4A). We then tested whether G2/M arrest by itself has a role in enhancing the radiosensitivity of these cells. Nocodazole, a drug that reversibly blocks microtubule polymerization, was used to accumulate A549 and H1299 cells in G2/M. The treatment schedule for Nocodazole (200 ng/ml) to induce the same degree of G2/M arrest compared with Ad-mda7 was 4 hours for A549 cells and 3.5 hours for H1299 cells. We then determined the radiosensitivity of A549 and H1299 cells treated with Nocodazole compared with controls, using clonogenic assays. The results indicate that G2/M arrest by itself, at least to the degree mediated by Ad-mda7, does not enhance the radiosensitivity of NSCLC cells (Fig. 4B). Ad-mda7 Enhances Radiosensitivity Independent of p53, Bax, and Fas We analyzed the expression of p53, Bax, and Fas protein in A549 and H1299 following treatment with either radiation alone, Ad-mda7 alone, Ad-mda7 plus radiation, AdLuc, or Ad-Luc plus radiation (Fig. 5A). The p53 protein levels dramatically increased in A549 cells treated with either radiation or Ad-mda7, but not with Ad-Luc. Fas protein expression in A549 cells treated with either radiation or Ad-mda7 was also increased and this effect was consistent
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FIG. 4. Ad-mda7 arrests cells in G2/M. (A) Cell cycle analysis of A549 and H1299 treated with either Admda7 or Nocodazole (200 ng/ml). The dose and exposure time of Nocodazole to accumulate the same proportion of cells in G2/M phase as was present 48 hours after Ad-mda7 transfection was determined in preliminary experiments. Data shown are representative of two independent experiments. (B) Clonogenic survival assays to determine radiosensitization by G2/M arrest induced by Nocodazole (200 ng/ml). Radiation was given after 4 hours of Nocodazole exposure for the A549 cell line, and after 3.5 hours of Nocodazole exposure for the H1299 cell line. Symbols represent radiation alone (filled diamond) and Nocodazole (open square). Bar: SE.
with a dependence on wild-type p53, as Fas protein expression in H1299 cells (which do not express p53) was not enhanced by these treatments. On the other hand, Bax protein expression was not significantly changed in either cell line by these treatments. Thus, because A549 and H1299 cells are equally radiosensitized by Ad-mda7, neither p53, Fas, or Bax correlated with radiosensitization by Ad-mda7. Ad-mda7 Enhances the Expression of p-c-Jun Protein It has been reported that radiation-induced apoptosis requires the activation of c-Jun N-terminal kinase (JNK) [15,16]. We tested whether Ad-mda7 was able to activate JNK and whether this correlated with radiosensitization. Rb, p-c-Jun, and JNK-1 protein levels were determined in A549, H1299, and CCD-16 cell lines treated with radiation alone, Ad-mda7 alone, Ad-mda7 plus radiation, Ad-Luc, or Ad-Luc plus radiation (Fig. 5B). Rb protein expression was reduced in A549 and H1299 cells treated with Ad-mda7, but was not changed with control vector in these cell lines. The expression of both p-c-Jun and JNK-1 was enhanced in A549 and H1299 cells treated with Ad-mda7. In CCD16 cells, however, Rb protein expression was slightly reduced, but expression of p-c-Jun and JNK-1 was not enhanced by Ad-mda7 treatment. These results are consistent with the possibility that Ad-mda7 mediates radiosensitivity and enhances apoptosis through the activation of JNK-1 and the subsequent activation of p-c-Jun.
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FIG. 5. Ad-mda7 enhances the expression of p-c-Jun but not Bax. (A) Western blot analysis of p53, Fas, and Bax protein expression in A549 and H1299 cells treated with either 5 Gy alone, Ad-mda7 alone, Ad-mda7 plus 5 Gy, Ad-Luc alone, or Ad-Luc plus 5 Gy. Cells were harvested 1 day after irradiation or 3 days after transfection. Vector concentrations used were identical as those used for Fig. 1. Actin was used as a loading control. (B) Western blot analysis of Rb, phosphorylated c-Jun, and MDA-7 protein in A549, H1299, and CCD16 cell lines treated with 5 Gy alone, Ad-mda7 alone, Ad-mda7 plus 5 Gy, AdLuc alone, or Ad-Luc plus 5 Gy. Cells were harvested 1 day after irradiation or 3 days after transfection. Vector concentrations used were identical as those used for Fig. 1. Actin was used as a loading control.
Curcumin Abrogates Ad-mda7 Mediated Radiosensitization Curcumin, a dietary pigment responsible for the yellow color of curry, has been reported to inhibit JNK activation [17]. We therefore determined the expression of p-c-Jun protein in A549 and H1299 cells treated with radiation alone, curcumin alone, Ad-mda7 alone, radiation plus curcumin, radiation plus Ad-mda7, or radiation plus curcumin plus Ad-mda7 (Fig. 6A). Curcumin when used alone enhanced p-c-Jun expression, as did Ad-mda7 used alone. However, curcumin reduced Ad-mda7 mediated activation of p-c-Jun in irradiated and unirradiated cells. To examine whether curcumin inhibits Ad-mda7 mediated radiosensitivity, we carried out clonogenic assays using A549 and H1299 lines. Cells were infected with Ad-mda7 and irradiated 48 hours later. Curcumin abrogated Ad-mda7 radiosensitization in both cell lines (Fig. 6B).
DISCUSSION We have compared the abilities of Ad-mda7 to radiosensitize NSCLC cell lines that have either wt-TP53 or deleted TP53 status and NHLF cell lines. The clonogenic survival results indicated that both the A549, which have wt-TP53,
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and the H1299, which is deleted for wt-TP53, cell lines were substantially radiosensitized by Ad-mda7 (Figs. 1A and 1B). However, two different NHLF cell lines were not (Figs. 1C and 1D). These results were consistent with the analysis of apoptosis by TUNEL (Fig. 2), suggesting that the radiosensitizing effect of Ad-mda7 in NSCLC cells involves a restoration of apoptosis propensity. Moreover, as both A549 and H1299 cells were equally affected, this activity of Ad-mda7 seems to be independent of the endogenous TP53 status of the cells. We also investigated whether this radiosensitizing effect of Ad-mda7 was due to an alteration of cell cycle phase distribution. Tumor cells are typically sensitive to radiation killing in the G2/M phase of the replicative cell cycle [18]. The transition from G2 to M is regulated in part by the mitosis-promoting factor, consisting of cyclin B1 and a G2-specific cyclin-dependent kinase Cdc2 [19]. Normally, cyclin B1 levels gradually increase as cells progress into the S and G2 phases of the cell cycle. As MDA-7 protein is expressed in these cell lines, the expression of cyclin B1 increases, peaking 2 days following Ad-mda7 transfection (Fig. 3), and the cells accumulate in G2/M (Fig. 4). Thus, there was the possibility that the radiosensitivity of the NSCLC lines might be enhanced solely by this accumulation in G2/M. We
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FIG. 6. Curcumin abrogates Ad-mda7 mediated radiosensitization. (A) Western blot analysis of p-c-Jun in A549 and H1299 cells treated with 5 Gy alone, curcumin (10 M), curcumin plus 5 Gy, Ad-mda7, Ad-mda7 plus 5 Gy, curcumin plus Ad-mda7, or curcumin plus Ad-mda7 plus 5 Gy. Cells were harvested 1 day after irradiation or 3 days after transfection. Curcumin was added 1 day after transfection. Actin was used as a loading control. (B) Clonogenic survival assays to determine radiosensitization in A549 and H1299 cells treated with either curcumin or curcumin plus Ad-mda7. Radiation was given 2 days after transfection. Curcumin was added 1 day after transfection. The vector concentrations used were identical to that used for Fig. 1. Each data point represents the average of three independent experiments. Bar: SE.
examined whether this degree of accumulation of G2/M phase has an important role in enhancing the radiosensitivity of these NSCLC lines by mimicking this effect with Nocodazole. Accumulating cells in G2/M with a dose of Nocodazole to a degree equal to or above that achieved with Ad-mda7 did not produce a radiosensitizing effect (Fig. 4B). These results indicate that the G2/M arrest produced by Ad-mda7 is not the principal cause of its radiosensitizing effect. We then investigated the expression of other proteins involved in apoptosis by western blot analysis. The expression of the pro-apoptotic protein Bax was not upregulated in either the A549 or H1299 cell lines, although the expression of p53 and Fas protein was increased in the A549 line, but not in H1299 (Fig. 5A). Thus, the changes in expression of these proteins do not correlate with the radiosensitizing effects of Ad-mda7. Other investigators have reported increased Bax protein after Ad-mda7 treatment of lung and breast cancer cells [9,13]. However, the dose of Ad-mda7 used in the present study was lower than previously described. In addition, Ad-mda7 has activity against Bax-deficient DU-145 cells, further suggesting that Bax upregulation is not a prerequisite for the effects of Admda7 [20].
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Rb is a negative regulator of apoptosis and suppresses the JNK/SAPK signaling pathway. This inhibition of JNK/SAPK activity may be an important mechanism by which Rb negatively regulates the stress-activated cellular events leading to cell death [21]. Rb has been shown to suppress radiationinduced apoptosis and, when Rb’s function is antagonized, apoptosis can proceed [22]. It is well established in the literature that activation of JNK is an important step in cells undergoing apoptosis in response to radiation [15,16]. Expression of Rb was suppressed in both A549 and H1299 cell lines following treatment with Ad-mda7, whereas JNK and c-Jun were concordantly activated (Fig. 5B). Based on these findings, it is possible that the activation of c-Jun and JNK-1 is mediated by the reduction of Rb protein expression induced by Ad-mda7. However, we also observed that JNK activation does not take place in the NHLF lines in spite of a reduction in Rb expression. Jiang et al. [10] have reported that Ad-mda7 suppresses the growth of cancer cell lines with defects in the Rb gene. Thus, activation of JNK and pc-Jun, but not suppression of Rb, correlates with the radiosensitizing effect of Ad-mda7. A role for activation of JNK in the radiosensitizing effect of Ad-mda7 was tested using curcumin, a non-specific inhibitor of JNK activation [17]. Curcumin suppressed the ability of Ad-mda7 to
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upregulate the expression of p-c-Jun in A549 and H1299 cells, consistent with an inhibition of JNK activity (Fig. 6A). Furthermore, neither cell line was radiosensitized when Admda7 was combined with curcumin (Fig. 6B). These data are consistent with, but do not prove, the possibility that Admda7 radiosensitizes NSCLC cells through activation of JNK and p-c-Jun. This appears to be blocked in normal cells, which are not radiosensitized by Ad-mda7. The mechanism by which JNK and p-c-Jun become activated by Ad-mda7 is not known, however, this may be due to a general activation of signal transduction pathways. Ad-mda7 has previously been shown to activate p38 [23] and PKR [24]. MDA7 protein also has been shown to activate STAT-3 [25]. In addition to two other vectors that we have previously examined, Ad-p53 and Ad-p16, Ad-mda7 has potent radiosensitizing properties. On A549 cells, Ad-mda7 seems to be a better radiosensitizer; Ad-mda7 produces a DRF of 1.93 compared with a DRF of 1.37 for Ad-p53 and a DRF of 1.75 for Ad-p16 [11,12]. This is further evidence to indicate that Ad-mda7 may radiosensitize through a novel mechanism that does not depend on p53. In light of this possibility, it would be of interest to test for an additive effect of the combination of Ad-p53 and Ad-mda7. This is currently under investigation. We have demonstrated that Ad-mda7 enhances the radiation sensitivity of NSCLC cells, but not of NHLF cells. Whether this occurs through activation of c-Jun has yet to be conclusively proven, but this possibility will be the subject of further studies. Our results indicate that the combination of Ad-mda7 and irradiation may be a useful gene therapy strategy for the treatment of NSCLC.
MATERIALS
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Cell culture, vectors, and chemicals. The human NSCLC cell lines, A549 (wt-TP53/wt-RB1) and H1299 (del-TP53/wt-RB1), and normal human lung fibroblast lines (NHLF), CCD-16 and MRC-9, were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained as specified by ATCC. The recombinant adenoviral vector (Ad-mda7) contains the CMV promoter, wild-type mda-7 cDNA, and an SV40 polyadenylation signal in a minigene cassette inserted into the E1-deleted region of modified Ad5. Adenovirus-mediated luciferase (Ad-Luc) was used as a control vector. These vectors have been described previously [6]. The preparations were tested for and determined to be free of replication-competent adenovirus and mycoplasma. Curcumin and Nocodazole were purchased from Sigma-Aldrich (Poole, UK). Stock solutions of curcumin (10 mM) were prepared freshly on the day of the experiment by dissolving the compound in ethanol. Mocktreated cells received the same concentration of ethanol. It was then diluted into medium at a concentration of 10 mM. Stock solutions of Nocodazole (5 mg/ml) were prepared by dissolving the compound in DMSO. It was then was diluted into medium (200 ng/ml). Gene delivery. In vitro transfection studies for all cell lines were performed by plating 2 ⫻ 105 cells in T25 flasks. Forty-eight hours after plating, cells were incubated for 1 hour with purified vector in 1 ml of medium without serum. After 1 hour, fresh medium supplemented with 10% FBS was added to the flask. Serum-free medium was used for mock transfection. Cells were further incubated for 48 hours before survival curves were generated.
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Radiation and clonogenic assay. Cells were irradiated with a high-dose rate 137Cs unit (3.7 Gy/minute) at room temperature in T25 flasks. Irradiation was performed 48 hours after vector treatment. The effectiveness of treatments was assessed by clonogenic assays. Briefly, monolayers of A549, H1299, CCD-16, and MRC-9 cells were treated in T25 flasks as described above, and after various doses of irradiation the cells were trypsinized and counted. Known numbers of cells were then replated in 100-mm culture dishes and returned to the incubator to allow macroscopic colony development. Colonies were counted after 10–14 days, and the percent plating efficiency and surviving fractions following given treatments were calculated based on the survival of nonirradiated cells treated with either mock infection, Ad-Luc or Ad-mda7. The vector treatments used were adjusted for each line to yield identical reductions in plating efficiency with Ad-mda7 (80%). The vector concentrations used, therefore, were 1000 vp/cell for A549, 250 vp/cell for H1299, and 1500 vp/cell for CCD-16 and MRC-9 cells. These treatments produced nearly 100% transfection efficiency. Some of the experiments on A549 cells used a different lot of Ad-mda7 vector which required 2000 vp/cell to achieve the same transfection efficiency. Apoptotic index and cell cycle analysis. Apoptosis was quantified by flow cytometry using the APO-BRDU Kit (Pharmingen, San Diego, CA). Briefly, 2 ⫻ 106 cells were fixed with 1% paraformaldehyde in PBS for 15 minutes at room temperature, washed twice with PBS, and stored in 70% ethanol at –20⬚C. For analysis, cells were incubated in the DNA labeling solution overnight at room temperature. Fluorescent labeled anti-BrdU antibody solution was added, and the cells incubated in the dark for 30 minutes at room temperature. The stained cells were analyzed by flow cytometry with the use of an EPICS flow cytometer (Coulter Corp., Hialeah, FL). All steps were performed according to the manufacturer’s recommendations. An analysis region was set based on the negative control, and the percentage of labeled cells was calculated from this region. We analyzed apoptotic indices 2 days after irradiation with 5 Gy or 4 days after infection. This time course was based on preliminary indications of the time for maximum apoptotic response. As before, infections with either Ad-mda7 or Ad-Luc were performed 48 hours before irradiation. Western analysis. Briefly, cells were scraped from the plates, washed with PBS, and lysed in cell lysis buffer. Protein (30 g) was electrophoretically separated on either 8% (for pRb), 12% (for Cyclin B1, pc-Jun, Fas, Bax and p53), or 15% (for MDA-7) SDS-polyacrylamide gels and transferred to polyvinylide difluoride membrane (Millipore, Bedford, MA). Mouse monoclonal antibodies for pRb, Cyclin B1 (Pharmingen, San Diego, CA), p53 (DAKO, Carpinteria, CA), and Fas (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal antibody for Bax (Santa Cruz Biotechnology, Santa Cruz, CA), MDA-7 (Introgen Therapeutics Inc., Houston, TX), JNK-1 (Promega, Madison, WI), were used as primary antibodies. Primary antibody for p-c-Jun, specific for cJun p39 phosphorylated on serine-63, was obtained from Santa Cruz Biotechnology. Primary antibody for JNK-1 detects the phosphorylated, active form of stress-activated protein kinase (SAPK), also known as cJun N terminal kinase, JNK. The membranes were enhanced by chemiluminescence using ECL western blot detection reagents (Amersham Corp, Arlington Heights, IL) according to the manufacturer’s instructions. Total cellular proteins applied to each lane were adjusted to equal concentration with BCA protein assay reagent (Bio-Rad Laboratories, Richmond, CA), and were confirmed with coomassie brilliant blue staining method.
ACKNOWLEDGMENTS This study was supported in part by grants R41 CA88421, P01 CA78778, P01 CA06294, and P30 CA16672 from the National Cancer Institute and a sponsored research agreement with Introgen Therapeutics, Inc. The vectors used in this research were the kind gift of Introgen Therapeutics, Inc. RECEIVED FOR PUBLICATION AUGUST 21, 2001; ACCEPTED SEPTEMBER 6, 2002.
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doi:10.1006/mthe.2002.0714, available online at http://www.idealibrary.com on IDEAL
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MOLECULAR THERAPY Vol. 6, No. 5, November 2002 Copyright © The American Society of Gene Therapy