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Departments of 1 Thoracic & Cardiovascular Surgery and 2 Biomathematics, University of Texas M. D. Anderson Cancer Center, Houston, Texas, and 3 Department of Internal Medicine and Pharmacology, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The FHIT gene encodes a protein composed of 147 amino acids and is a member of the histidine triad (HIT) nucleotide-binding protein superfamily (3) . Several lines of experimental evidence, both in vitro and in vivo, have supported the tumor suppressor role of the FHIT gene. For example, the exogenous expression of FHIT protein in FHIT-deficient human cancer cells inhibited tumor cell proliferation in vitro (4 , 5) and suppressed tumor growth and tumorigenicity in vivo (4 , 6) by altering the cell cycle and inducing apoptosis. In addition, using an FHIT transgenic mouse model, Fong et al. (7) recently noted that the FHIT-heterozygous (+/–) mice developed multiple visceral and skin tumors similar to those seen in patients with Torre’s syndrome, which is caused by a deficiency in a mismatch repair gene. Conversely, the reintroduction of the wild-type FHIT gene into the FHIT-deficient mice prevented tumor development (8) . The molecular mechanism involved in the FHIT-mediated tumor-suppressing activities remains to be elucidated, however.
The tumor suppressor gene p53, on the other hand, is a well-established cellular gatekeeper that plays an important role in the regulation of numerous biological processes, including cell proliferation, cell cycle progression, apoptosis, and tumor surveillance (9) . The p53 gene is also the most frequently mutated gene in human cancers (10) with >50% of NSCLCs processing a mutation in this gene (11) . Transfer of the wild-type p53 gene, on the other hand, has proved effective in suppressing the proliferation of tumor cells bearing mutated p53 as seen in vitro, in animal models, and in patients with cancer (12, 13, 14, 15) . However, because many cancer cells, including lung cancer cells, express wild-type p53 and most tumors are also heterogeneous with respect to their p53 status, p53 gene transfer alone may be insufficient to suppress tumor cell growth because of the general resistance of the wild-type p53-expressing tumor cells to p53 gene transfer (12 , 14 , 16) .
Although the efficiency of p53 in preventing cell proliferation is a strong deterrent to malignant progression, the activity of p53 is tightly regulated by divergent extracellular and intracellular signals through the mechanisms that result in degradation, stabilization, or accumulation of p53 protein (9 , 17) . One protein that plays an essential role in the regulation of p53 is the MDM2 protein, which functions as a ubiquitin ligase for p53 (17, 18, 19) . Multiple cellular pathways also exist in the regulation of MDM2 activity (17, 18, 19) . One of the mechanisms that are potentially responsible for the resistance of wild-type p53-expressing tumors to p53 gene transfer may be imposed via a negative feedback pathway of p53 and MDM2 in which the introduction of exogenous p53 induces the overexpression of endogenous MDM2, which, in turn, results in rapid degradation of the p53 protein in the ubiquitin-proteasome system (20 , 21) .
We and others (4 , 5) have studied the effects of FHIT and p53 on tumor cell proliferation and apoptosis in the context of diverse biological activities, especially the ability of FHIT to induce apoptosis and alter cell cycle kinetics in various types of cells and the apparent link of the FHIT genomic aberrations to the integrity of p53 function in lung tumorigenesis (22) . In this study, we used the adenoviral vector-mediated cotransfer of FHIT and p53 in NSCLC cells with a varying p53 status to evaluate the interaction and therapeutic potential of FHIT and p53 gene coexpression by using isobologram modeling. We also sought to elucidate the molecular mechanism involved in the tumor suppression activities mediated by the interaction between FHIT and p53 proteins in vitro and in vivo. We present here the first evidence that the coexpression of FHIT and p53 synergistically inhibited tumor cell proliferation in both p53-sensitive and p53-resistant NSCLC cell lines in vitro and suppressed the growth of p53-resistant tumor xenografts in nude mice. We also demonstrated that the synergism of the FHIT- and p53-mediated tumor-suppressing activity was associated with the FHIT-mediated inactivation of MDM2 and the subsequent stabilization of the p53 protein. Our results provide insight into the interaction of FHIT and p53 and the FHIT-mediated inhibition of tumor cell growth and regulation of p53 activity and suggest novel strategies for cancer gene therapy.
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MATERIALS AND METHODS |
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Adenoviral and Plasmid Vectors.
Recombinant adenoviral vectors Ad-p53 and Ad-FHIT, which contain the wild-type p53 gene and an FHIT gene, respectively, were used as gene therapy agents, and either Ad-LacZ, which contains a ß-galactosidase gene, or Ad-GFP was used as a nonspecific negative control. Construction of these recombinant adenoviral vectors has been described previously (4)
. Viral stocks were prepared by the Vector Core Facility at The University of Texas M. D. Anderson Cancer Center (Houston, TX). Viral titers were determined by absorbance measurements (viral particles/ml) and plaque assays (plaque forming units/ml). Potential contamination of the viral preparations by a wild-type virus was monitored by PCR analysis. Expression plasmid vectors containing the cDNA of FHIT, HDM2, LacZ, and GFP genes were used to transfect NSCLC cells using a FuGENE 6 transfect reagent (Roche Molecular Biochemicals, Indianapolis, IN).
Immunofluorescence Staining.
Cells were first cultured in chamber slides and treated with adenoviral vectors at various multiplicities of infection (MOI) for 24 h. Cells were then fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min on ice. Cells were rinsed twice with PBS and permeabilized with 0.2% Triton X-100 for 10 min. For immunostaining, cells were incubated with rabbit anti-FHIT (Zymed Laboratories, South San Francisco, CA) and mouse anti-p53 or anti-MDM2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies diluted in PBS containing 5% BSA for 1 h at room temperature. FITC-labeled antirabbit IgGs and rhodamine-labeled antimouse IgGs (Chemicon International, Temecula, CA) were diluted 1:200 in PBS, and the cells were incubated with the antibodies for 30 min. The nuclei were stained by 4',6-diamidino-2-phenylindole and then examined under an Eclipse E400 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a Sensys digital camera (Photometrics, Tucson, AZ) and Metamophore software (Universal Imaging Corp., Downingtown, PA).
Growth Inhibition and 2,3-Bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-Tetrazolium-5-Carboxanilide Inner Salt Assay.
Inhibition of tumor cell growth by adenoviral vector-mediated transfer of FHIT and p53 genes was analyzed by quantitatively determining cell viability using an improved 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay (Roche Molecular Biochemicals). PBS was used as a mock control and Ad-LacZ as a negative control. Briefly, cells were plated in 96-well microtiter plates at 1 x 103 cells/well in 100 µl of medium. One day after the cells were plated, 25-µl aliquots of medium containing adenoviral vectors at various MOI (viral particles/cell) were added. Cells were then incubated at 37°C in a humidified atmosphere under 5% CO2. Four days after transduction, cell viability was quantified in a microplate reader (Model MRX; Dynatech Laboratories, Chantilly, VA) by a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay according to the manufacturer’s instructions. The percentage of viable cells was calculated in terms of the absorbency in treated cells relative to the absorbency in untreated control cells. Experiments were repeated at least three times and triplicate samples were used for each treatment.
Isobologram Analysis of the Interaction of FHIT and p53.
The effects of the coexpression of two tumor suppressor genes, FHIT and p53, on tumor cell growth in five human cell lines (WI-38, H1299, H460, H322, and A549) were analyzed quantitatively and statistically in vitro by the improved isobologram method of Steel and Peckham (23)
. The mathematical operation and statistical analysis methods have been described elsewhere (23
, 24)
. The MOI for the cotransduction were based on the IC50 values determined from each individual vector in different cell lines (Table 1)
. An equal amount of viral particles from each of the two vectors in cotransduction experiment is used to bring the total viral particles to the same amount as those in a single vector transduction.
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. Seventy-two h after transduction, cells were collected, fixed in 4% paraformaldehyde, permeabilized with 70% ethanol, washed with PBS, and stained with propidium iodide solution containing 40 µg/ml propidium iodide and 10 µg/ml DNase-free RNase A. DNA fragmentation was analyzed by flow cytometry. The percentage of apoptotic cells was shown by the cells in the G0-G1 phase.
Immunoprecipitation, Immunodepletion, and Western Blot Analysis.
For the preparation of crude cell lysates, cells were suspended in immunoprecipitation SDS-PAGE running buffer (radioimmunoprecipitation assay) containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and a complete set of proteinase inhibitors (Roche Molecular Biochemicals) and lysed for 20 min at 4°C. Cell lysates were passed through a 25-gauge needle and briefly sonicated twice for 30 s each. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). For the immunoprecipitation studies, cell lysates were precleared by incubation with 10 µl of protein A/G-agarose (Santa Cruz Biotechnology, Inc.) for 30 min at 4°C and then centrifugation. Each protein sample (500 µg) was incubated with its respective antibody at 4°C overnight, followed by incubation with 25 µl of A/G-agarose beads. After centrifugation, the resultant supernatant was saved as an immunodepleted fraction. The precipitated beads were then washed three times in radioimmunoprecipitation assay buffer and once in PBS. The bound proteins were solubilized by adding 25 µl of SDS-containing sample buffer. The crude cell lysates and the immunoprecipitated and immunodepleted samples were used in standard SDS-PAGE and Western blot analyses.
Quantitative Real-time Reverse Transcription-PCR (RT-PCR).
The TaqMan probe and primers for the MDM2 gene were designed using Primerexpress software (Perkin-Elmer Applied Biosystems, Foster City, CA) and synthesized by the same manufacturer. The human genomic DNA or total RNAs were used as template standards, and the human ß-actin or glyceraldehyde-3-phosphate dehydrogenase TaqMan probes and primers were used as their respective internal controls. Total RNAs were isolated from Ad-FHIT- and Ad-p53-transduced tumor cells using Trizol reagent (Life Technologies, Inc., Grand Island, NY), as instructed by the manufacturer, and RNA samples were treated with DNase (Life Technologies, Inc.). Real-time RT-PCR and the quantification of RT-PCR products were performed and the products analyzed using a TaqMan Gold RT-PCR Kit, an ABI Prism 7700 Sequence Detection System, and the appropriate software according to the manufacturer’s instructions (Perkin-Elmer Applied Biosystems).
Efficacy of Combination Treatment with Ad-FHIT and Ad-p53 in Animal Models.
All animals were maintained and animal experiments performed according to NIH and institutional guidelines established for the Animal Core Facility at M. D. Anderson. The animals used in this study were female Nu/nu mice (6–8 weeks of age) that were purchased from Charles River Laboratories (Wilmington, MA). Before tumor cell inoculation, mice were subjected to 3.5 Gy of total body irradiation from a 137Cs radiation source.
A549 cells were used to establish s.c. tumors in mice. Briefly, 1 x 106 cells were injected into the right flank of each mouse. When the average size of the xenograft tumors reached 5–8 mm in diameter, the mice were randomly divided into six treatment groups: PBS control; single treatments with Ad-FHIT, Ad-p53, or Ad-LacZ vector alone; and combination treatment with Ad-p53 + Ad-FHIT and Ad-p53 + Ad-LacZ. Each treatment group contained eight mice, and experiments were repeated twice.
Mice were treated according to the following schedule: on day 0, each mouse was injected intratumorally with adenoviral vector (Ad-p53, Ad-FHIT, or Ad-LacZ) at a dose of 3 x 1010 viral particles/tumor in a volume of 0.2 ml. Ear tags were placed on the mice so that data obtained from individual animals could be traced. Tumor dimensions were measured three times/week using a digital caliper.
Statistical and Mathematical Analyses.
Tumor volume was calculated using the equation 
, where a is the largest diameter, and b is the smallest diameter. Differences in tumor volumes between treatment groups were analyzed using the ANOVA test and statistical software. A difference was considered to be statistically significant when P 
0.05.
A mathematical model and a statistical method were developed to analyze the effects of combination treatments in our animal model according to the improved isobologram method of Steel and Peckham (23, 24, 25) Tumor volumes (V) were fitted with dose-response curves fa(Da) for reagent A and fb(Db) for reagent B by the following equations: V = fa(Da) and V = fb(Db). Then mode I, mode IIa, and mode IIb could be expressed as follows: VMode I = fa(Da) x fb(Db); VMode IIa = fb[fb–1fa(Da) + Db]; and VMode IIb = fa[fa–1fb(Db) + Da]. The range of additivity was defined as being between the maximum (Vmax) and minimum (Vmin) tumor volume among these three isoeffect volumes: VMode I, VMode IIa, and VMode IIb. When the observed tumor volume after combination treatment was between Vmax (boundary between addition and antagonism) and Vmin (boundary between addition and synergism), the treatment combination effect was regarded as additive. When the observed tumor volume after combination treatment was below Vmin or above Vmax, the effect was regarded as synergistic or antagonistic, respectively. The Wilcoxon signed rank test was used for statistical analysis, and P < 0.05 was taken to indicate a significant difference.
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RESULTS |
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. In particular, the fluorescence images showed nuclear staining (4',6-diamidino-2-phenylindole, blue) and cytoplasmic and nuclear staining for FHIT (FITC, green) and p53 (rhodamine, red) in H1299 cells (Fig. 1A, a–d)
and A549 cells (Fig. 1A, e–h)
24 h after the cotransduction of Ad-p53 and Ad-FHIT. Specifically, the coexpression of FHIT and p53 was seen in 97.6% of H1299 cells and 95.4% of A549 cells. 
2 statistical analysis showed that these coincidences of the expression of both proteins in both types of cells were significant (P < 0.001), indicating that equivalent levels of expression of both the FHIT and p53 proteins could be achieved in transduced cells.
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The dose effect of each agent on tumor cell proliferation was then subjected to the median-effect equation to generate dose-response curves (24)
. The ID50 values (doses of adenoviral vectors that inhibited cell growth by 50%) were then determined on the basis of the resultant dose-response curves (Table 1)
. The responses of NSCLC cell lines to Ad-p53 or Ad-FHIT varied from the most sensitive (low ID50) to the most resistant (high ID50) in the following orders of sensitivity: H1299 > H322 > A549 > H460 (Table 1)
. Although Ad-LacZ had a detectable inhibitory effect on cell proliferation, the effect was significantly less than that of Ad-FHIT or Ad-p53, and ID50 values of Ad-LacZ could be determined only at a very high dose (Table 1)
, supporting the specificity of FHIT- and p53-mediated tumor suppression activities.
The combined effects of FHIT and p53 on cell proliferation were evaluated by isobolograms generated (Fig. 1B)
using averaged data from experiments done independently at least twice for all cell lines tested. Combination treatment with Ad-FHIT + Ad-p53 exhibited a synergistic antiproliferative effect in all cancer cell lines tested independent of their p53 status, whereas no synergistic effect was observed in normal human lung fibroblasts WI-38 cells (Fig. 1B)
. On the other hand, the combination of either Ad-p53 + Ad-LacZ (Fig. 1B
, middle panels) or Ad-FHIT + Ad-LacZ (Fig. 1B
, bottom panels) showed no synergistic effects in any of the cancer cell lines. A nonparametric statistical analysis of the predicted data versus the observed data showed that the observed synergistic effects on tumor cell proliferation of Ad-FHIT + Ad-p53 were statistically significant in all cancer cell lines (H1299, P = 0.0117; H322, P = 0.0181; H460, P = 0.0051; A549, P = 0.0117; Table 1
).
Effects of Coexpression of FHIT and p53 on Apoptosis.
One of the hallmark molecular events induced by tumor suppressors is apoptosis. To study the combined effects of FHIT and p53 on apoptosis, suboptimal doses (slightly lower than the ID50 values in each NSCLC line shown in Table 1
) of Ad-FHIT and Ad-p53 vectors were applied to each cancer cell line. For the combination treatment, the ratio of the MOI (viral particles/cell) of Ad-FHIT to Ad-p53 was 500:50 in the H1299 cells and 2500:2500 in the H460, normal human bronchial epithelial, and WI-38 cells. For the single treatment, either Ad-FHIT or Ad-p53, an appropriate amount of Ad-LacZ vectors, was added to make the total viral particles equal to that of the combination treatment.
The apoptosis and cell-cycle kinetics in cells transduced with Ad-FHIT and Ad-p53 were then analyzed by flow cytometry in conjunction with propidium iodide staining (Fig. 1C)
. The accumulation of cells in the sub-G0-G1 phase analyzed with propidium iodide staining was correlated with positive cells analyzed with terminal deoxynucleotidyl transferase-mediated nick end labeling staining by flow cytometry. A low level of apoptosis was observed in the H1299 and H460 cells transduced with Ad-FHIT alone at 72 h after posttranduction (Fig. 1C)
. An intermediate level was seen in the cells transduced with Ad-p53 alone (Fig. 1C)
. However, a supra-additive induction of apoptosis was seen in these same cell lines when they were cotransduced with Ad-FHIT and Ad-p53, but no significant enhancement of apoptosis was observed in normal cell lines cotransduced with Ad-FHIT and Ad-p53 at the same viral doses and at the same time after transduction (Fig. 1C)
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Consistent with the flow cytometry results, caspase-9 was also found to be activated in the H1299 and H460 cells transduced with either Ad-FHIT or Ad-p53 alone or in combination, as shown by the cleavage products detected by Western blot analysis (Fig. 1D)
. In addition, caspase-9 was strongly activated in H460 cells (wild-type p53) in which the endogenous p53 was activated by cisplatin treatment followed by the transduction with Ad-FHIT (Fig. 1D
, Lane F+CP) as compared with the effects in cells transduced with Ad-GFP (Fig. 1D
, Lane G+CP). These results suggest that the observed synergism in the inhibition of tumor cell proliferation produced by the coexpression of FHIT and p53 is due to a synergistic induction of apoptosis.
Synergistic Inhibition of Tumor Growth by the Coadministration of Ad-p53 and Ad-FHIT in Vivo.
To determine whether the synergistic growth inhibition mediated by the cotransfer of the FHIT and p53 genes observed in vitro could be reproduced in vivo, we evaluated the combined effects of FHIT and p53 overexpression on tumor growth by directly coadministering Ad-FHIT and Ad-p53 vectors into human A549 s.c. xenografts in nude mice. Mice were divided into six treatment groups: four groups that received single treatment with PBS; Ad-LacZ, Ad-p53, or Ad-FHIT alone; and combinations of Ad-p53 + Ad-FHIT or Ad-p53 + Ad-LacZ. Each treatment group contained five to eight mice, and all experiments were repeated twice. The overall effects of treatments on tumor growth were analyzed by an ANOVA statistical method. Treatment with Ad-p53 + Ad-FHIT significantly inhibited tumor growth in the A549 tumor model (P < 0.05) in comparison with the results seen in the other treatment and the control groups (Fig. 2A)
. We also analyzed whether there was a synergistic interaction between the combination treatment elements in these tumors using a modified isobologram method (see "Materials and Methods"). This showed that coadministration of Ad-p53 and Ad-FHIT produced a significant synergistic inhibitory effect on tumor growth in the A549 tumor model (P < 0.05; Fig. 2B
).
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. The overexpression of FHIT up-regulated the expression of endogenous wild-type p53 proteins >6-fold in Ad-FHIT-transduced H460 and A549 cells but not in the H322 cells with endogenous mutant p53 (Fig. 3A)
. A modest increase of exogenous p53 was also seen in Ad-p53 and Ad-FHIT cotransduced cells, independent of the endogenous p53 status in these cells (Fig. 3B)
. However, the level of the exogenous FHIT protein was not affected by either the endogenous or exogenous expression of p53 (Fig. 3A
, middle panels), suggesting that the synergistic effect of the tumor suppressor genes may be due to FHIT-mediated up-regulation of the p53 protein but not the reverse.
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, PBS). By comparison, in the presence of exogenous Ad-FHIT, the endogenous p53 protein in these cells could still be detected even 24 h after cyclohexamide treatment (Fig. 3C
, Ad-FHIT). These results indicate that expression of FHIT protein indeed stabilizes the p53 protein.
Regulation of the MDM2 by Coexpression of FHIT and p53.
There are multiple pathways to stabilize p53 in response to different forms of stress. One of the key pathways is to inhibit MDM2-mediated p53 degradation by dissociating MDM2 from p53 (21
, 26)
. To determine whether the enhanced stability of p53 is associated with the inactivated MDM2 in NSCLC cells cotransduced with Ad-FHIT and Ad-p53, we analyzed the expression of MDM2 proteins by Western blot analysis using a monoclonal MDM2 antibody (SMP-14; Santa Cruz Biotechnology, Inc.; Fig. 4A
). We detected significantly decreased level of the 90-kDa MDM2 protein in the cells cotransduced with Ad-FHIT and Ad-p53 in comparison with control cells treated with Ad-p53 alone or with Ad-p53 + Ad-LacZ (Fig. 4A)
in all cell lines tested. Similar results were observed when MDM2 antibodies from different sources were used (N-20 from Santa Cruz Biotechnology, Inc., and IF2 from Oncogene, Cambridge, MA; data not shown).
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. Transcription of the MDM2 mRNA was significantly up-regulated in both the Ad-p53-transduced and Ad-FHIT + Ad-p53-cotransduced cells but not in the cells transduced with Ad-FHIT alone (Fig. 4B)
. These results suggest that the overexpression of p53 activates MDM2 at the transcriptional level through a feedback mechanism but that the FHIT-mediated regulation of MDM2 expression occurs at the posttranscriptional protein level.
To exclude the possibility that the adenoviral vector and p53 expression might have affected MDM2 expression, we analyzed the effect of FHIT expression on exogenous MDM2 expression in p53-null H1299 cells cotransfected with the HDM2 and FHIT expressing plasmid vectors (pHDM2 and pFHIT). H1299 cells cotransfected with pHDM2 and pLacZ were used as the control. Consistent with the results obtained in the adenoviral vector-mediated FHIT gene transfer experiment (Fig. 4A)
, the level of exogenous MDM2 protein was also reduced in the cells cotransfected with pHDM2 and pFHIT plasmids, compared with the levels in cells cotransfected with pHDM2 and pLacZ (Fig. 4C)
. Furthermore, MDM2 proteins were clearly detected at a similar rate of recovery after treatment with a broad proteasome inhibitor, MG132, in all treatment groups (Fig. 4C)
, suggesting that the FHIT-mediated reduction in the MDM2 protein is independent of the proteasome degradation pathway.
Interruption of Association of MDM2 with p53 by FHIT.
To further our understanding of the mechanism behind the observed stabilization of the p53 protein by FHIT, we investigated the interactions among the FHIT, MDM2 and p53 proteins in Ad-FHIT- and Ad-p53-transduced wild-type p53-bearing H460 and A549 cells using immunoprecipitation and Western blot analysis (Fig. 5)
. The FHIT protein was detected in MDM2-immunoprecipitated complexes in both H460 (Fig. 5A
, Lane 2) and A549 (Fig. 5A
, Lane 6) cells transduced with FHIT, indicating a direct interaction between the FHIT and MDM2 proteins. In addition, a significantly smaller amount of the FHIT/MDM2 complexes was observed in both H460 (Fig. 5A
, Lane 4) and A549 (Fig. 5A
, Lane 8) cells that were cotransduced with Ad-FHIT + Ad-p53. These results suggest that FHIT may cause degradation of MDM2 as implied by the reduced MDM2 levels in Ad-FHIT + Ad-p53-transduced cells (Fig. 4A)
and that a large amount of p53 proteins may also interfere with the apparent FHIT-MDM2 interaction.
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. We found that MDM2-bound p53 was detected in the MDM2-immunoprecipitated complexes in both H460 (Fig. 5B
, Lane 1) and A549 (Fig. 5B
, Lane 3) cells transduced with Ap-p53. However, the levels of MDM2-bound p53 protein were dramatically reduced in both H460 (Fig. 5B
, Lane 2) and A549 (Fig. 5B
, Lane 4) cells cotransduced with Ad-FHIT + Ad-p53 vectors compared with the levels in cells transduced with Ad-p53 alone. Furthermore, Western blot analysis of p53 proteins in MDM2-immunodepleted crude protein fractions showed that almost all of the p53 protein was associated with MDM2 in the absence of FHIT expression (Fig. 5B
, Lanes 5 and 7), but the MDM2-p53 protein interaction was significantly interrupted by the presence of FHIT coexpression (Fig. 5B
, Lanes 6 and 8). These results strongly suggest that the interaction of FHIT with MDM2 effectively blocks the association of MDM2 with p53, and this prevented the MDM2-mediated degradation thus enhancing the stability of p53 protein.
We also performed immunofluorescence image analysis of FHIT and MDM2 protein expression to study the interaction of these two proteins in living A549 cells in which endogenous p53 expression was induced by cisplatin and exogenous FHIT expression was induced by Ad-FHIT (Fig. 5C)
. In the Ad-FHIT-transduced cells (Fig. 5C, a and b)
, the FHIT protein was detected mainly in the cytosol (Fig. 5C, a)
, and the MDM2 protein was predominantly located in the nucleus (Fig. 5C, b)
. In untransduced cells (Fig. 5C, b)
, MDM2 protein expression could also be detected in the cytosol, but this was not seen in the FHIT-expressing cells (Fig. 5C, b)
. In addition, the fluorescence intensity of the MDM2 protein was reduced in nuclei in the FHIT-expressing cells compared with the intensity in the non-FHIT-expressing cells, indicating that a FHIT-mediated reduction in MDM2 protein expression and a FHIT-mediated interference in the association of MDM2 with p53 occurred in living cells.
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DISCUSSION |
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There is increasing evidence that the inactivation of multiple tumor suppressor genes has a synergistic effect on tumor development and proliferation (22 , 27) . For example, >50% of NSCLCs have been found to carry p53 mutations (11) and 50–70% of NSCLCs also have deficient FHIT gene expression (28 , 29) . In addition, several studies have demonstrated that an allelic imbalance within the FHIT locus frequently coexists with p53 abnormalities and that this may be an early event in NSCLC pathogenesis (30 , 31) . From a clinical perspective, NSCLCs are highly resistant to conventional treatments such as surgery, radiotherapy, and chemotherapy (32) . On the basis of these observations and known facts, combination treatment with synergistic tumor-suppressing gene therapy (in our case, adenoviral vector-mediated FHIT and p53 gene cotransfer) may constitute a rational and effective strategy for the treatment of a wide range of both lung and other cancers.
The stability of p53 is the key to the maintenance of multiple cellular functions such as cell cycle arrest and apoptosis. The p53 tumor suppressor gene is activated in response to diverse cellular stresses such as those inflicted by DNA-damaging agents and to oncogenic signals through mechanisms that result in the stabilization and accumulation of wild-type p53 protein (20
, 33) . Overexpression of FHIT protein up-regulated both the endogenous and exogenous wild-type p53 expression in NSCLC cells transduced by the Ad-FHIT vector alone or cotransduced by Ad-FHIT and Ad-p53. However, the latter results in synergistically enhanced tumor suppression and apoptotic activities, which may be a direct reflection of the FHIT-mediated stabilization of p53. This implicates an important molecular pathway in the regulation of FHIT-mediated tumor-suppressing activity. Although the sensitivity of tumor cells to the adenoviral vector-mediated FHIT transfer-induced growth inhibition was not significantly correlated with the p53 gene status in the cells tested (4
, 6)
, the activation of endogenous p53 in cells possessing wild-type p53 by a chemotherapeutic agent such as cisplatin or the induction of the exogenous expression of p53 in p53-null or mutant cells by a gene therapy agent such as Ad-p53 could be used to enhance the therapeutic efficacy of Ad-FHIT in a wide range of tumor cells. For example, as shown in this study, in the H460 cells with wild-type p53 pretreated with cisplatin and then transduced with Ad-FHIT, the expression of endogenous p53 was induced, the FHIT-mediated stabilization of the p53 protein was massively enhanced, and caspase-9 was activated (Fig. 1D)
. Of further relevance, the induction of apoptosis mediated by Ad-p53 and Ad-FHIT also follows a different time course. That is, a peak apoptotic induction is seen for Ad-p53 at 
48 h and for Ad-FHIT at 96 h after transduction (33
, 34) . They also induce apoptosis via different pathways (35)
. These differences in therapeutic kinetics and molecular function may thus also contribute to the observed synergistic effect of combination treatment with Ad-FHIT and Ad-p53 on both growth inhibition and apoptosis.
Structural and functional analysis of the MDM2 protein has revealed that MDM2 interacts directly with p53 in the NH2-terminal domains (20 , 36) . As a result of this interaction, MDM2 directly blocks the transcription factor and a tumor suppressor activity of p53 (37) and targets p53 for degradation by proteolysis (33 , 38) . It has thus become clear that one way to stabilize and activate p53 in cells is by interrupting the interaction of MDM2 and p53 (39) . Ways to do this are suggested by the fact that the activity of MDM2 is also regulated by covalent modifications and by noncovalent regulators, both of which can modulate the ability of MDM2 to bind p53 (19) . In particular, two tumor suppressor proteins, human p16Ink4A and murine p19Arf (human p14Arf), have been shown to bind to MDM2 and inhibit the MDM2-mediated degradation of p53 (40 , 41) . We observed a significant reduction of MDM2 proteins in cells cotransduced by Ad-FHIT and Ad-p53 compared with those transduced by Ad-p53 alone. We also noticed a significant reduction of MDM2-p53 complexes and detected the direct MDM2-FHIT interaction in those cells. These results suggest that the interaction of FHIT with MDM2 may interfere the association of MDM2 with p53 and, subsequently, interrupt MDM2-medaited p53 degradation. For some reason, however, the effect was evident only in the Ad-p53 and Ad-FHIT-cotransduced cells. Thus, the synergy in FHIT and p53-mediated suppression of tumor growth and induction of apoptosis may be related to the FHIT-stabilized high level of p53 in these cells. FHIT may induce apoptosis by another mechanism in cells with endogenous wild-type p53.
Our data suggest that the FHIT-mediated reduction in MDM2 expression is neither controlled by the down-regulation of MDM2 mRNA transcription nor by the activation of the proteasome-dependent MDM2 degradation pathway. It also appears that the p14Arf protein is not involved in the FHIT-mediated regulation of MDM2 expression because both H460 and A549 cell lines express no p14Arf protein as a result of a homozygous gene deletion. However, the phosphorylation of MDM2 and the splice isoform expression of MDM2 may regulate MDM2 protein function such that it serves as an E3 enzyme that degrades the p53 protein (42 , 43) . We examined these possibilities using Western blot analysis, which showed that a different migrating form of the phosphorylated MDM2 protein occurs in the presence of anti-phospho (Ser166)-MDM2 antibody in a vector-dose-dependent manner. We also detected different possible splicing isoforms of MDM2 proteins under nonreduced conditions in Ad-FHIT-treated cells (data not shown). Although more detailed studies are needed to clarify and confirm these findings, we propose that the overexpression of FHIT probably inactivates the MDM2 protein by inducing a change in the phosphorylation status of the MDM2 protein and promoting cleavage of the protein into a nonfunctional species.
Taken together, our results indicate that the coexpression of the FHIT and p53 tumor suppressor genes promotes a synergistic inhibition of tumor cell growth in vitro and in vivo. We also identified a novel molecular mechanism for this FHIT-mediated tumor suppression and conclude that FHIT is important to the regulation of p53 because of its ability to interact with other cellular p53 regulators such as MDM2. Our study also points the way for development of novel strategies for cancer gene therapy that involve the combined delivery of two or more tumor suppressor genes that can synergistically induce apoptosis in cancer cells with a defined genotype.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: M. Nishizaki and J. Sasaki contributed equally to this work
Requests for reprints: Lin Ji, Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Unit 445, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-1443; Fax: (713) 794-4901; E-mail: lji{at}mdanderson.org
Received 1/20/04. Revised 5/ 4/04. Accepted 6/11/04.
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. If the observed data are distributed inside, above, or below the envelope of additivity, the interaction is considered additive, antagonistic, or synergistic, respectively. The interaction of the gene therapy combination (Ad-p53 + Ad-FHIT, Ad-p53 + Ad-LacZ, and Ad-FHIT + Ad-LacZ) in each cell line is demonstrated by individual isobolograms. Statistical analysis was performed using the Wilcoxon signed rank test. P 
and the dashed line with 
, respectively. Results are reported as the mean of relative tumor volumes. The inhibition of tumor growth by combination treatment with Ad-p53 + Ad-FHIT is significantly synergistic (P = 0.0033).
