Material and methods

Materials

DOTAP and cholesterol were purchased from Avanti Polar Lipids (Albaster, AL). RPMI-1640 medium, Ham's/F12 medium and fetal bovine serum (FBS) were purchased from GIBCO-BRL-Life Technologies (New York, NY). Rabbit anti-FUS1 poylclonal antibody was developed against a synthetic oligopeptide (gasgskarglwpfasaa) derived from the N-terminal amino-acid sequence of the FUS1 protein (Bethyl Laboratories, Montgomery, TX) and used for immunohistochemical analysis and Western blotting.¹⁵

Cell lines and animals

Human non-small-cell lung carcinoma (NSCLC) cell line H1299 was a gift from Drs Adi Gazdar and John D Minna (UT Southwestern Medical Center, Dallas, TX). A549 cells were purchased from American Type Culture Collection (Manssas, VA). H1299 and A549 cells were maintained in RPMI-1640 and Hams-F12 medium, respectively, that were supplemented with 10% FBS, 1% glutamate and antibiotics. Cells were regularly passaged and tested for presence of mycoplasma. Female BALB/c nude (nu/nu) mice (Charles River Laboratories, Wilmington, MA), 4–6 weeks old, used in the study were maintained in a pathogen-free environment and handled according to the institutional guidelines established for animal care and use.

Cloning and purification of plasmids

FUS1 cDNA and CAT cDNA was subcloned into the multiple cloning site of pVAX plasmid DNA vector containing kanamycin resistance marker (Invitrogen Inc. Carlsbad, CA). Additional vector developed in the laboratory included the pLJ143/KGB2/FUS1 plasmid vector that consisted of a CMV minimum promoter with an E1 enhancer at the 3' end, a BGH-poly A signal sequence at the 5' end, kanamycin-resistance gene, and a minimum pMB1 origin of replication (ori) sequence. The presence of the appropriate inserts was confirmed by DNA sequence analysis and by restriction enzyme analysis. The plasmids were subsequently transformed into Escherichia coli. DH5- strain (Stratagene, Carlsbad, CA) and were purified as described previously.^{16, 17}

Synthesis of liposomes and preparation of DNA:liposome complex

An amount of 20 mM DOTAP:Chol liposome was synthesized and extruded through Whatman Filters (Kent, UK) of decreasing size (1.0, 0.45, 0.2, and 0.1 mm) as described previously.^{17, 18} For preparation of DNA:liposome complexes DOTAP:Chol (20 mM) stock solution and DNA solution diluted in 5% dextrose in water (D5W) were mixed in equal volumes and mixed to give a final concentration of 4 mM DOTAP:Chol–150 g DNA in 300 l final volume (ratio 1:2.6) as described previously.¹⁷ The DNA:liposome mixture thus prepared was used in all the experiments described in the present study.

Measurement of particle size analysis

Freshly prepared DNA:liposome complexes were analyzed for mean particle size using the N4 particle size analyzer (Coulter, Miami, FL). The average particle size of the DNA:liposome complexes ranged between 375 and 400 nm.

In vitro transfection and transgene expression

Human NSCLC cells (H1299, and A549) were seeded in six-well plates at 5 10⁵ cells/well for Western blot analysis and 10⁵ cells/well in two-well chamber slides for immunocytochemical analysis. The following day, cells were transfected with DOTAP:Chol–FUS1 complex (2.5 g DNA) in serum-free medium for 3 hours. Following transfection, cells were replenished with complete medium and incubated at 37°C. For Western blot analysis, cells were harvested at 24 and 48 hours post transfection and analyzed for FUS1 protein expression. For immunocytochemical analysis, cells were fixed in 1% glutaraldehyde and stained as described previously.¹⁹ FUS1 expression was detected using a polyclonal rabbit anti-human FUS1 polyclonal antibody.

Effect of DOTAP:Chol–FUS1 complex on subcutaneous lung tumor xenograft

Prior to the start of the experiment mice were irradiated (3.5 Gy) using a cesium source to enhance tumor uptake. Briefly, female nu/nu mice were injected with 5 10⁶ H1299 cell and A549 cells into the lower right flank. Tumor formation in mice was observed twice or three times weekly. Treatment was initiated when the tumors were 50–100 mm³ in size (day 0). Animals were divided into groups (n=10/group) and treated with FUS1 plasmid DNA, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex. Animals were treated daily by intratumoral injections for a total of six doses (50 g DNA/dose). Tumors were measured every 2 or 3 days by using calipers. The tumor volume was calculated as described previously.¹⁷ Animals were euthanized by CO₂ inhalation when the tumor had reached 1.5 cm³ in size or was necrotic.

Effect of DOTAP:Chol–FUS1 complex on experimental lung metastasis

To establish lung metastasis, female nu/nu mice were injected intravenously via tail vein with 1 10⁶ A549 tumor cells suspended in 200 l of sterile PBS. After 6 days, mice were divided into groups (n=10/group) and treated as follows: treated with PBS, treated with plasmid DNA alone, treated with liposome alone, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex. Mice were treated with 10 g DNA:liposome complex intravenously (i.v.) via tail vein using a 27-gauge needle daily for a total of six doses. Animals were euthanized by CO₂ inhalation 3-weeks following the last dose. Lungs from each of the mice from the five groups were injected intratracheally with India ink and fixed in Feketes solution. The therapeutic effect of each groups were determined by counting the number of metastatic tumor nodules in each lung under a dissecting microscope without knowledge of the treatment groups. The data were analyzed and interpreted as statistically significant if the P-value was <.05 by the Mann–Whitney rank-sum test.

Animal survival experiments

To determine the therapeutic effect of DOTAP:Chol–FUS1 complex on animal survival female nu/nu mice were injected intravenously via tail vein with 1 10⁶ A549 tumor cells suspended in 200 l of sterile PBS. After 6 days, mice were divided into groups (n=5/group) and treated as follows: treated with PBS, treated with plasmid DNA alone, treated with liposome alone, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex. Mice were treated daily by i.v. injections of the liposome:DNA complex (10 g DNA) via the tail vein using a 27-gauge needle. Animals received a total of six doses (10 g DNA/dose). Animals were monitored daily for morbidity and mortality. Animals that were moribund were euthanized by CO₂ inhalation. The therapeutic effects of the treatments were determined by statistical analysis using the Kaplan–Meier survival estimation and Wilcoxon signed-rank sum tests.

Terminal deoxynucleotide transferase (TdT)-mediated biotin uridine triphosphate nick-end labeling (TUNEL) assay for DNA fragmentation

To determine the fate of tumor cell following treatments, subcutaneous tumor tissues harvested from animals that received various treatments were subjected to TUNEL staining as described previously.¹⁹ Slides were counterstained with 0.4% methyl green. In all the staining procedures, appropriate negative controls were included. Stained tissue sections was observed under microscopy and the number of TUNEL positive cells, an indicator of apoptotic cells, were determined by semiquantitative analysis as described elsewhere.¹⁹

Statistical analysis

The statistical significance of the experimental results was calculated using the Whitney rank-sum test for tumor measurements and lung metastases, and the Wilcoxon log-rank test and Kaplan–Meier survival test for animal survival.

Results

In vitro expression of FUS1 protein

Prior to testing the therapeutic effect of FUS1 gene in vivo, transgene expression of the FUS1 plasmid DNA complexed to DOTAP:Chol liposome was tested in vitro. Transfection of lung tumor cells, H1299 and A549 resulted in detection of FUS1 protein at 24 h and 48 h (Fig 1a). Furthermore, FUS1 protein was localized in the cytoplasm and perinucleus of the tumor cells as demonstrated by immunohistochemical analysis (Fig 1b).

Figure 1.

Expression of FUS-1 in lung cancer cells. Human non-small-cell lung cancer cells (H1299) were transfected with DOTAP:Chol–FUS1 complex (2.5 g DNA) and analyzed for FUS1 expression using an anti-FUS1 monoclonal antibody. (a) Western blot analysis demonstrated FUS1 expression on days 1, 2 and 3 after transfection. (b) Immunohistochemical analysis revealed cell membrane, cytoplasm, and perinuclear staining for FUS1. Hoechst staining indicates the nucleus of the cell.

Full figure and legend (138K)

Intratumoral injection of DOTAP:Chol–FUS1 complex suppresses tumor growth

We tested the tumor suppressor function of FUS1 gene in vivo by direct intratumoral injection of DOTAP:Chol–FUS1 complex. Treatment of subcutaneous lung tumor xenograft (H1299, and A549) for a total of six doses with DOTAP:Chol–FUS1 complex resulted in a significant suppression of tumor growth (P=.005 for H1299, and P=.01 for A549) compared to control animals that were treated with FUS1 plasmid DNA, and treated with DOTAP:Chol–CAT complex (Fig 2).

Figure 2.

Inhibition of subcutanous lung tumor xenograts by FUS1. Subcutaneous lung tumor xenografts were established by injecting H1299 and A549 tumor cells (5 10⁶) in nude mice. Animals were randomly divided into groups (n=10/group) and treated with FUS1 plasmid, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex. Animals were treated when tumors were 50–100 mm³ (day 0) daily for a total of six doses (50 g DNA/dose) and tumor growth monitored. A significant inhibition of tumor growth was observed in both, H1299 (P=.005) and A549 (P=.01) tumor-bearing mice treated with DOTAP:Chol–FUS1 complex. In contrast, no significant growth inhibition was observed in control animals that were treated with PBS and treated with DOTAP:Chol–CAT complex. Error bars denote standard error.

Full figure and legend (28K)

The fact that the tumor inhibition was due to FUS1 protein expression was demonstrated by immunohistochemical analysis. FUS1 protein expression was detected in the subcutaneous tumor tissues primarily localized to the cytoplasm as observed in vitro (data not shown). Expression was primarily observed in the tumor cells. However, expression in other cells intermixed with tumor cells were also observed. The subtype of cells staining positive for FUS1 in the tumor was not determined. Furthermore, tumors treated with DOTAP:Chol–FUS1 complex underwent significant apoptotic cell death as evidenced by TUNEL staining compared to tumors from those animals that were treated with plasmid DNA or DOTAP:Chol–CAT complex (Fig 3). Induction of apoptotic cell death was observed in both H1299 and A549 tumors.

Figure 3.

Induction of apoptosis in subcutanous tumors treated with DOTAP:Chol–FUS1. Subcutaneous lung tumor (H1299 and A549) xenografts that were treated with FUS1 plasmid, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex were harvested and subjected to TUNEL staining. Animals were treated DOTAP:Chol–FUS1 complex demonstrated 15–35% TUNEL-positive staining of tumor tissues (33% for H1299 and 18% for A549; P=.001). In contrast, no significant growth inhibition was observed in control animals that were treated with PBS and treated with DOTAP:Chol–CAT complex. Error bars denote standard error.

Full figure and legend (40K)

Intravenous injection of DOTAP:Chol–FUS1 complex inhibits experimental lung metastasis

To test the tumor suppressor activity of FUS1 on experimental lung metastases, lung tumors were established by injecting A549 tumor cells via tail vein. Intravenous treatments of these lung tumor bearing animals with DOTAP:Chol–FUS1 complex resulted in a significant inhibition (P=.001) of lung metastases compared to control animals that were treated with PBS, treated with FUS1 plasmid DNA, treated with liposome alone, and treated with DOTAP:Chol–CAT complex (Fig 4). Animals treated with DOTAP:Chol–CAT complex demonstrated some tumor inhibition. The ability of DOTAP:Chol–CAT complex treatments to demonstrate some antitumor activity is not surprising and is attributed to nonspecific antitumor activity. However, tumor inhibition was not significant compared to animals treated with PBS, treated with FUS1 plasmid DNA and treated with liposome. These results show that the therapeutic effect observed in lung tumor-bearing animals when treated with DOTAP:Chol–wtFUS1 is specific to wtFUS1.

Figure 4.

DOTAP:Chol–FUS1 complex inhibits experimental lung metastasis. Experimental lung metastasis was established in nude mice by injecting A549 tumor cells (10⁶) via tail vein. After 6 days, animals were divided randomly into groups (n=10/group) and treated with PBS, treated with plasmid DNA alone (10 g), treated with liposome alone, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex. Animals were treated daily for a total of six doses (10 g DNA/dose) via tail vein. Animals were euthanized 3-weeks after the last treatment; lungs were injected with India ink solution and harvested. Lungs were examined under a stereomicroscope and the number of tumor nodules counted. A significant (P=.001) reduction in the number of tumor nodules was observed in animals treated with DOTAP:Chol–FUS1 complex compared to control animals. Error bars denote standard error.

Full figure and legend (48K)

Intravenous treatment of lung tumor-bearing animals with DOTAP:Chol–FUS1 complex prolongs animal survival

We next evaluated the effect of DOTAP:Chol–FUS1 treatments on animal survival. Treatment of experimental A549 lung tumor-bearing animals with DOTAP:Chol–FUS1 complex resulted in a significant (P=.01) and prolonged survival (mean survival time=80 days) (Fig 5). In contrast, no significant survival of animals was observed that were treated with PBS (mean=47.8), treated with FUS1 plasmid (mean=51.6), treated with liposome (mean=47.2), and treated with DOTAP:Chol–CAT complex (mean=47.8). Furthermore, histopathological analysis of various organs demonstrated no significant treatment-related toxicity.

Figure 5.

Treatment with DOTAP:Chol–FUS1 complex increases animal survival. Experimental lung metastasis was established in nude mice by injecting A549 tumor cells (10⁶) via tail vein. After 6 days, animals were divided randomly into groups (n=5/group) and treated with PBS, treated with plasmid DNA alone (10 g), treated with liposome alone, treated with DOTAP:Chol–CAT complex, and treated with DOTAP:Chol–FUS1 complex. Animals were treated daily for a total of six doses (10 g DNA/dose) via tail vein. Following the last treatment, animals were monitored daily for morbidity and mortality. Survival was estimated by using the Kaplan–Meier and Wilcoxon signed-rank tests. Survival was significantly longer in animals treated with DOTAP:Chol–FUS1 complex (mean survival time: 80 days; P=.01) compared to animals that were treated with PBS (mean survival time: 47.8 days), treated with plasmid DNA alone (mean survival time: 51.6 days) treated with liposome alone (mean survival time: 47.2 days), and treated with DOTAP:Chol–CAT complex (mean survival time: 47.8 days).

Full figure and legend (19K)

Discussion

We have previously demonstrated DOTAP:Chol liposome effectively deliver tumor suppressor genes, p53 and Fhit, to lungs and inhibited lung tumor growth.¹⁷ However, recent studies indicate that alterations in the p53 and Fhit gene occur in the later stages of lung cancer development.⁷ Thus, restoration of a gene that is altered early in tumor progression may be a better therapeutic for lung cancer treatment. Recently, identification of nine genes in a narrow 120-kb region on chromosome 3p21.3 has been reported.¹² FUS1 is one among the nine genes located in this region. However, the tumor suppressor functions of each of the nine genes have not been well studied. In the present study, we therefore tested the tumor suppressor activity of FUS1 gene in human lung cancer cells.

Treatment of subcutaneous and experimental lung metastasis with FUS1 gene resulted in significant suppression of tumor growth with induction of apoptosis. The ability of FUS1 to induce apoptosis and inhibit tumor growth is similar to findings reported by Ji et al.¹⁴ That the observed tumor suppression was due to FUS1 expression was demonstrated by immunohistochemistry. Although the exact function(s) of FUS1 gene is not known, localization of the FUS1 protein to the cell membrane, cytoplasm and perinucleus suggest that it may have role in signal transduction. More recently, myristolyation of FUS1 protein as a requirement for tumor-suppressive activity was demonstrated.¹⁵ However, further characterization of this gene is essential to elucidate its function.

To further evaluate the effects of systemic delivery of FUS1 on experimental lung metastasis, lung tumor-bearing animals were treated with liposome–FUS1 complex. A significant inhibition of lung metastatic tumor nodules was observed in animals treated with liposome–FUS1 DNA complex. The tumor inhibitory effect by FUS1 was comparable to that observed when treated with p53.¹⁷ However, unlike our previous study, we used lower concentrations of plasmid DNA in the present study and still achieved the same therapeutic effect. These results suggest two possibilities, one that FUS1 may be more potent than p53 and two, use of lower concentrations of plasmid DNA will result in achieving the same therapeutic effect. Preliminary results indicate that FUS1 is more potent than p53 (data not shown). Supporting this observation is the study by Ji et al.^{14, 15} In their study, treatment with Ad-FUS1 inhibited tumor metastases more effectively than Ad-p53. Use of lower concentrations of plasmid DNA also will minimize the DNA associated inflammatory response resulting in reduced toxicity and therefore be of advantage when used in clinical trials. However, further detailed comparative studies are warranted to justify these observations. The nonspecific antitumor activity observed with the CAT DNA is not surprising and is in agreement with previous reports where nonspecific antitumor activity was demonstrated when treated with control plasmid DNA's complexed to liposomal vectors.^{17, 20, 21, 22, 23} The induction of an inflammatory response following injection of liposome–DNA complex has been shown to mediate the nonspecific activity antitumor activity.^{20, 21} However, the antitumor activity observed in animals treated with wt-FUS1 was significantly higher than in animals treated with CAT demonstrating the specificity.

The effect of FUS1 treatments on animal survival was next evaluated. Treatment with DOTAP:Chol–FUS1 complex significantly prolonged survival of A549 lung tumor-bearing animals. At the time of writing this report, 40% of the tumor-bearing mice treated with DOTAP:Chol–FUS1 complex were still alive (125 days) indicating that tumors were either eliminated or their growth delayed. Whatever the reason may be, it is clear that FUS1 can suppress tumor growth and prolong survival.

Although we have demonstrated FUS1 has potent tumor suppressive activity, the functions of some of the other genes located in the 120-kb region have not been studied. Ji et al¹⁴ recently demonstrated antitumor activity for three of the nine genes. Thus, treatment of lung cancer with a combination of these genes may result in a more potent therapeutic effect than that observed with individual genes. Additionally, combination of DOTAP:Chol–FUS1 treatment with other treatment strategies such as chemotherapy may result in a more effective tumor suppression and is of clinical relevance. These possibilities are currently under investigation in our laboratory.

In conclusion, we have demonstrated the tumor suppressive activity of FUS1 gene and that intratumoral and systemic delivery of FUS1 gene is a potential therapeutic strategy for the treatment of early preneoplastic lesions as well as in the treatment of localized and disseminated lung tumors.

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Acknowledgements

We thank Nora Rios for assistance in preparation of the manuscript. This work was supported in part by Public Health Service Grant P01 CA78778-01A1 (JAR), by the Specialized Program of Research Excellence (SPORE) in Lung Cancer 2P50-CA70970-04 (JDM and JAR); by a Career Development Award P50-CA70907-5 (RR); by gifts to the Division of Surgery, from Tenneco and Exxon for the Core Laboratory Facility; by the UT MD Anderson Cancer Center Support Core Grant CA 16672; by the Texas Tobacco Settlement Fund as appropriated by the Texas State Legislature (Project 8), by the MD Anderson WM Keck Center for Cancer Gene Therapy (JR, RR), by Texas Higher Education Coordinating Board ATP/ARP Grant 003657-0078-2001 (RR); by BESCT Lung Cancer Program grant DAMD17-01-1-0689 (LJ, RR); by TARGET Lung Cancer Grant DAMD17-02-1-0706 [LJ, RR]; by Cancer Center Support (CORE) Grant CA 16672; and by a sponsored research agreement with Introgen Therapeutics, Inc.