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Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [L. J., B. F., N. Y., J. A. R.], and Hamon Center for Therapeutic Oncology Research and Departments of Internal Medicine and Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-8593 [K. F., J. D. M.]
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Introduction |
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Experimental evidence is needed to clarify the function of FHIT as a tumor suppressor gene. If loss of FHIT function leads to human cancers, one might predict that replacement of the abnormal FHIT gene with the wild-type gene would result in tumor suppression similar to that shown for the Rb or p53 tumor suppressor genes, including inhibition of cell growth in vitro and suppression of tumorigenicity and tumor growth in vivo. Siprashvili et al. (14) recently showed that replacement of the wild-type FHIT gene in human cancer cell lines that lacked endogenous Fhit expression significantly reduced their tumorigenicity in nude mice. In contrast, Otterson et al. (15) recently reported that introduction of wild-type FHIT into HeLa cells, a cervical carcinoma cell line, did not alter its tumorigenicity in animals. To date, studies of the function of FHIT as a tumor suppressor gene have been performed in tumor cell lines with transient or stable transfection by wild-type FHIT-expressing plasmids. Thus, we decided to use an adenoviral vector as an alternative means for studying the tumor suppressor function of FHIT. We studied the effects of FHIT gene overexpression mediated by recombinant Ad vector (Ad-FHIT) on cell proliferation, apoptosis, and cell cycle kinetics in human lung and head and neck cancer cells with varied status of FHIT gene or gene products and on tumorigenicity and tumor growth in nude mice.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Construction of Recombinant Ads.
The recombinant Ad-CMV-FHIT-GAL4 (Ad-FHIT) was constructed using a cosmid-Ad vector system by homologous recombination in recA+ bacteria, Escherichia coli. The FHIT cDNA (568 bp) was placed in an expression cassette containing a CMV promoter and bovine growth hormone (BGH) poly(A) sequence. The CMV-FHIT-BGH poly(A) expression cassette was inserted into the E1-deleted region in an Ad vector containing an inactivated E4 region. A vector expressing GFP, Ad-CMV-GFP (Ad-GFP), was used to monitor transduction efficiency by the viral vectors and as a nonspecific transgene expression control. Ad-E1--GAL4 (Ad-EV), an empty E1- vector containing the same E4 modification as in the Ad-FHIT vector, was used as a negative control. These viral vectors were amplified in either 293 cells or 293/VP16 cells and purified with the conventional CsCl gradient centrifugation procedure, as described previously (16
, 17)
. Viral titers were determined by both absorbance measurement and plaque assay for pfu. Potential contamination of the viral preparation by the wild-type virus was monitored by PCR analysis.
Determination of Cell Growth Rate.
Inhibition of cell growth by Ad transduction was assayed by MTT staining of viable cells (Sigma Chemical Co., St. Louis, MO). In brief, cells were transduced with adenoviral vectors at varied MOI by directly applying the diluted vectors into the growth medium. The experimentally treated cells were harvested at a designated time, and 200 µl of cell suspension was added to each well in 96-well plates. A one-tenth volume of MTT solution (5 mg MTT/ml PBS) was added to each well, and each plate was incubated for 2–4 h at 37°C until a purple precipitate was visible. The medium was then carefully removed, and precipitates were dissolved in 100 µl of DMSO. Growth rate was plotted as the percentage of viable cells in PBS-treated controls (a value arbitrarily set at 100%). Each experiment was repeated at least three times with each treatment given in duplicate or triplicate. Data were presented as an average of the results from individual experiments.
Western Blot Analysis.
Expression of FHIT in Ad-FHIT-transduced cells or untransduced (PBS-treated) controls was analyzed by Western blotting. Cells grown in 6-well plates (1–2 x 106/well) were treated with adenoviral vectors or with PBS alone as a control. Cells were transduced with Ad-FHIT in vitro. At designated time points, cells were harvested by centrifugation for 5 min at 2500 rpm at 4°C. Cells were washed two times with cold PBS and then resuspended in 100 µl of cell lysis solution [20 mM HEPES (pH 7.9), 150 mM NaCl, 0.5 mM DTT, 10 mM KCl, 0.2 mM EDTA, 25% glycerol, and complete proteinase inhibitors (Boehringer Mannheim, Indianapolis, IN)]. Cells were sonicated with two pulses of 30 s each at a setting of 6.0 (MICROSON; Heat Systems Inc., Farmingdale, NY). The sonicated cells were then spun for 10 min at 14,000 rpm at 4°C. Cell lysates were collected and stored at -80°C. Protein concentrations of the cell lysates were determined using Bio-Rad protein assay reagent (Bio-Rad, Fremont, CA). Proteins were then separated by SDS-PAGE. For electrophoresis, each lane was loaded with about 60 µg of crude proteins and run at 100 V for 1–2 h. Proteins were then transferred from gels to Hybond-ECL membranes (Amersham International, Little Chalfont, England). Membranes were blocked in a blocking solution (3% dry milk and 0.1% Tween 20 in PBS) for 1 h at room temperature. Membranes were then incubated with a 1:1000 dilution of primary rabbit antihuman Fhit fusion protein polyclonal antiserum (Zymed Inc., South San Francisco, CA) and a 1:1000 dilution of mouse anti-ß-actin monoclonal antibodies (Amersham International). Immunocomplexes were detected with the secondary horseradish peroxidase-labeled rabbit antimouse IgG or goat antirabbit IgG antibodies using an enhanced chemiluminescence kit (Amersham International), according to the manufacturer’s instructions.
Apoptosis and Cell Cycle Analysis.
Apoptosis was analyzed by FACS using the TUNEL reaction with FITC-labeled dUTP (Boehringer Mannheim Biochemicals, Mannheim, Germany). In brief, cells (1 x 106/well) were seeded on 6-well plates and infected with Ad constructs. PBS containing no viral constructs was used as a control. Cells (including floating cells in the medium) were harvested at designated times and washed twice with PBS. Cells were fixed in 1% formaldehyde for 15 min at 4°C, permeabilized in 500 µl of 70% ethanol, and stored at 4°C. Cells were then washed twice with PBS, resuspended in 50 µl of TUNEL reaction mixture [0.2 M potassium cacodylate, 25 mM Tris-HCl (pH 6.6), 2.5 mM cobalt chloride, 0.25 mg/ml BSA, 100 units/ml terminal deoxynucleotidyltransferase, and 10 µM FITC-labeled dUTP], and incubated for 60 min at 37°C in a humidified atmosphere in the dark. After incubation, cells were washed twice in PBS and resuspended in 500 µl of PI solution (5 µg/ml PI and 10 µg/ml Rnase A in PBS). Cells were analyzed for green FITC, fluorescence through a 520-nm BP filter, and for red (PI), fluorescence through a 620-nm LP filter. Green fluorescence was taken as a marker of DNA fragmentation and apoptosis; red fluorescence, as a marker of DNA content and cell-cycle status.
Animal Studies.
All animals were maintained, and all animal experiments were conducted under institutional guidelines established for the Animal Core Facility at M. D. Anderson Cancer Center. Nu/Nu mice were obtained from Charles River (Cambridge, MA). Five mice were used for each treatment group. For the tumorigenicity assay, H1299 cells were transduced in vitro with Ad-FHIT at a MOI of 10, with PBS alone as a mock control and Ad-EV as a negative control. The transduced cells were harvested at 24 h and 48 h after transduction, respectively. Viability of cells was determined by trypan blue exclusion staining. Viable cells (1 x 107) were then injected s.c. into the right flank of female nude mice, 6–8 weeks of age. Tumor formation in mice was observed twice or three times weekly up to 3 months. Tumor dimensions were measured every 2 or 3 days by using a linear caliper. The tumor volume was calculated using the equation 
, where a is the largest dimension and b is the perpendicular diameter.
To study the effect of FHIT on tumor growth, H1299 cells were used to establish s.c. tumors in nude mice. Briefly, 1 x 107 cells were injected into the right flank of female nude mice, 6–8 weeks of age. When the tumors reached 5–10 mm in diameter (at about 2 weeks postinjection), animals received intratumoral injections of Ad-FHIT or control vectors, respectively, for four times at days 1, 4, 8, and 11, at a total dose of 3 x 1010 pfu/tumor. Tumor size was measured and calculated as described above. At the end of the experiment, animals were killed and tumors were excised and processed for pathological and immunohistochemical analysis.
Statistics.
Both the in vitro and the in vivo results were expressed as mean ± SD or mean ± SE. Student’s two-sided t test was used to compare the values of the test and control samples. A value of P < 0.05 was taken as significant.
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Results |
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. Cells from each line were transduced in vitro by Ad-FHIT vectors administered at various MOI; cells treated with PBS, empty vector Ad-EV, and Ad-GFP were used as controls. The transduction efficiency was determined by examining the GFP-expressing cells in the Ad-GFP-transduced cell population under a fluorescence microscope. The transduction efficiency of the adenoviral vectors was >80% at the highest MOI applied for each cell line. Cell proliferation was analyzed by determining the viability of cells at 1, 2, 5, and 5 days after transduction, respectively. Viability was significantly reduced in Ad-FHIT- transduced H1299 cells that exhibit deletion of exon 5 and a 120-bp insertion in the FHIT gene (Ref. 9
; Fig. 1A
), A549 cells that exhibit deletion of exon 4 and fusion of exon 3 and exon 5 (Ref. 7
; Fig. 1B
), H460 cells that exhibit deletions of exons 5, 6, and 7 (Ref. 9
; Fig. 1C
), and 1483 cells that exhibit deletion of exon 4 (Ref. 18
; Fig. 1D
). In all cases, the viability of transduced cells was compared with that of untransduced (PBS-treated) control cells (the viability of which was set at 100%). Fewer than 20% of cells transduced by Ad-FHIT at the highest MOI survived 5 and 7 days after transduction when compared with the PBS mock–treated controls, whereas the growth of cells transduced by the Ad-EV and Ad-GFP control vectors at the same MOI was only slightly affected. In contrast, adenoviral vector-mediated FHIT expression minimally affected cell growth in 22B cells (Fig. 1E)
, a head and neck carcinoma cell line containing and expressing the normal FHIT gene. Nearly 80% of cells transduced by Ad-FHIT, at a MOI of 20, were still viable 7 days after transduction.
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. A <10% loss of cell viability was observed in Ad-FHIT-transduced cells at various MOI when compared with that in untransduced control cells, and similar losses were also observed in Ad-EV-transduced cells throughout the posttransduction time course.
The effects of FHIT on cell proliferation in Ad-FHIT-transduced cells were consistent with the levels of exogenous FHIT gene expression and the status of the endogenous FHIT gene in these cell lines, as demonstrated by Western blot analysis using polyclonal rabbit antihuman Fhit antibodies as probes (Fig. 2)
. Overexpression of the Fhit protein was detected in all Ad-FHIT-transduced cells, but not in untransduced controls. In addition, the levels of Fhit expression increased with increasing MOI and with days after transduction in all cell lines examined (Fig. 2
, Lanes 4– 15). No endogenous expression of Fhit was detected in H1299, A549, H460, and 1483 tumor cells (Fig. 2, A-D
, Lanes 1–3), which have been shown to lack the FHIT gene and to form aberrant FHIT transcripts. Endogenous Fhit protein expression was detectable at low levels in both normal FHIT transcript-expressing 22B cells (Fig. 2C)
and normal HBEC cells (Fig. 2D)
. Together, these data indicated that the Ad-FHIT vector efficiently mediated FHIT gene expression in these transduced tumor cells and that the expressed FHIT proteins could inhibit tumor cell proliferation and growth in vitro.
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. Induction of apoptosis was detected in Ad-FHIT-transduced cells. More than 20% and 35% of cells were apoptotic at days 5 and 7 after transduction with Ad-FHIT, respectively, whereas only about 7% and 10% of cells treated with PBS alone and transduced with Ad-EV vector, respectively, were TUNEL positive at the same time periods. The level of induction of apoptosis in the Ad-FHIT-transduced cells increased with time after transduction and correlated with the expression of Fhit protein (Fig. 2)
and the viability of cells (Fig. 1)
, suggesting that the inhibition of tumor cell proliferation by Fhit is mediated directly or indirectly through induction of apoptosis.
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. Interestingly, cell cycle processes in the Ad-FHIT-transduced cells seemed to be significantly affected by overexpression of Fhit at later stages of transduction compared with the untransduced and Ad-EV-transduced controls. Cell cycle profiles indicated that >32% of the H1299 cells transduced with Ad-FHIT were arrested at S phase at 5 days posttransduction (Fig. 3B, I)
, whereas only 8% of untransduced control cells (Fig. 3B, G)
and 9% of Ad-EV-transduced cells (Fig. 3B, H)
were arrested at S phase. This phenomenon had not been observed in any previously reported in vitro experiment using FHIT-expressing plasmids.
Inhibition of Tumorigenicity by Ad-FHIT-transduced Cancer Cells.
To further determine whether the observed inhibitory effects of FHIT on tumor cell proliferation in vitro could be demonstrated in vivo, we tested the tumorigenic potential of Ad-FHIT-transduced H1299 cells in nude mice (Fig. 4)
. Viable H1299 cells (1 x 107/mouse) transduced with Ad-FHIT for 24 h (Fig. 4A)
or 48 h (Fig. 4B)
were s.c. injected into five mice in each treatment group. Tumors started to form around 10 days after injection and were observed in all mice who received injections of either untreated or Ad-EV-transduced H1299 cells. Once tumors were established, they grew aggressively, ultimately reaching an average volume of about 0.5–0.9 cm3 in mice who received injections of untreated cells and 0.4–0.7 cm3 in mice who received injections of 24-h and 48-h Ad-EV transductants within 5 weeks after injection, respectively. In contrast, however, no tumors formed in any mice injected with Ad-FHIT-transduced H1299 cells within the same periods of time (Fig. 4, A and B)
, indicating that the tumorigenicity of Ad-FHIT-transduced H1299 cells was eliminated in vivo.
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and until 24 days after last injection for the A549 tumor model (Fig. 5B)
, respectively. All of the tumors in the mice treated with Ad-FHIT showed significantly suppressed growth compared with tumors in PBS-treated and Ad-EV-treated mice in both tumor models (P < 0.001; Fig. 5, A and B
). At the termination of these experiments, the tumors injected with Ad-FHIT were, on average, only about one-tenth the size of the tumors injected with PBS alone or with Ad-EV vector.
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Discussion |
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The lung cancer cell lines H1299, A549, and H460 and the head and neck carcinoma cell line 1483 all exhibit an altered or inactivated FHIT gene, as shown by reverse transcription-PCR and Northern blot analysis (7 , 9 , 18) , lack endogenous Fhit protein expression, as shown by Western blot analysis, and are highly tumorigenic. Alterations in the FHIT locus have been shown to be correlated with loss or reduction of Fhit protein expression in tumors and tumor cell lines (3 , 15 , 19 , 20) . Clonal expansion of Fhit-negative cells has been observed in many primary and cultured tumors (14 , 19) . These data suggest that the lack of Fhit expression might provide a selective advantage for clonal expansion in vivo and implicate FHIT gene in cancer pathogenesis. Moreover, frequent loss of heterozygosity at the 3p14 region and the absence of Fhit protein expression have been observed in precancerous lesions (19 , 21) ; both have also been associated with the G1 morphological grade and early clinical stage in renal carcinoma (20) . Together, these data suggest the inactivation of FHIT in the early phases of cell carcinogenesis and pathogenesis. Our observations that exogenous wild-type FHIT gene replacement inhibited growth in these cancer cell lines provide supporting evidence for the theory that functional inactivation of FHIT participates in cancer pathogenesis and are consistent with a role for FHIT as a tumor suppressor gene. On the other hand, overexpression of exogenous Fhit did not affect growth of head and neck carcinoma 22B cells and normal HBECs, which express both normal FHIT transcripts and Fhit protein. Similar results have also been observed by Siprashvili et al. (14) in Ad type 5-transformed human kidney 293 cells expressing endogenous Fhit protein. A high yield of colonies of FHIT transfectants and, in our case, production of Fhit-expressing recombinant Ads could be achieved in this cell line. These results suggest that exogenous Fhit functions specifically in restoring inactivated endogenous FHIT alleles and that it does not cause generalized toxicity.
In this study, we have demonstrated for the first time that the growth inhibition of tumor cells is associated with the induced apo-ptosis and altered cell cycle processes resulting from overexpression of exogenous Fhit. The decreased cell proliferation is consistent with the increased apoptotic activity and the increased level of exogenous Fhit protein expression in Ad-FHIT-transduced tumor cells, suggesting that the inhibition of tumor cell growth by Fhit is mediated by an apoptotic pathway. Increased apoptosis in association with altered cell proliferation and inhibited cell growth has been described in various systems (22, 23, 24, 25) . However, the effects on cell proliferation, apoptosis, and cell cycle processes demonstrated by Fhit seem different from those of the tumor suppressor genes p53 and Rb. The cellular responses to the expression of exogenous Fhit protein in terms of both growth inhibition and apoptosis induction are slower than those to the overexpression of exogenous p53, although the magnitude of the final effect is similar (24 , 25) . In our study, overexpression of Fhit proteins was detected at 24 h after transduction, but the apparent growth inhibition and apoptosis induction were observed first at 3 days and peaked at 4–5 days after transduction. By comparison, however, the growth inhibition and apoptosis induced by exogenous p53 overexpression became apparent at 24 h and peaked at 72 h after transduction in the same cell lines (17 , 25 , 26) . Moreover, Ad-FHIT-transduced cells accumulate in S phase, whereas p53-overexpressing cells (22 , 23) and Rb1-positive cells responding to effectors such as p16 overexpression undergo G0-G1 arrest (27) . These differences suggest that an apoptotic pathway mediated by the FHIT gene is different from the pathways mediated by the tumor suppressor genes p53 and Rb. It is likely that FHIT-mediated apoptosis is p53-independent because it can occur in cells homozygously deleted for p53 (H1299).
The mechanism of induction of apoptosis and alteration of cell cycle processes by Fhit is not yet well understood, but a few clues are emerging from recent structural and functional studies of Fhit protein and its homologues in the HIT protein superfamily (26) . Crystallography showed that binding nucleotide substrates by Fhit, similar to G proteins, changed the molecular surface of the protein and, thus, suggested that a Fhit-substrate complex might be the active form of Fhit involved in the signaling pathway(s) controlling cell growth (28, 29, 30, 31) . Ap4A and Ap3A, which are substrates for the Fhit hydrolase, have emerged as putative intracellular and extracellular signaling molecules involved in mediation of adaptive responses by mammalian cells to environmental stress such as heat, oxidation, and DNA damage (1 , 30 , 32) . Furthermore, it has recently been reported that apoptosis in cultured human cells is associated with alterations in Ap3A and Ap4A levels (33 , 34) . These data suggest that the loss of FHIT gene function could result in alterations in both the levels of substrates and the status of substrate-binding, which, consequently, could inactivate a pathway that leads to apoptosis and initiate the malignant process by stimulating DNA synthesis and cell proliferation. Discovery of the direct target of Fhit-substrate complexes in the cell proliferation, cell cycle progression, and apoptosis cascade would be expected to provide insights into the molecular mechanisms of Fhit function and could undoubtedly shed light on molecular targets or therapeutic agents for the prevention and treatment of various human cancers associated with the functional inactivation of the FHIT gene.
In our study, overexpression of FHIT efficiently eliminated the tumorigenicity of the highly tumorigenic cell line H1299 in vivo. Although one might argue that the diminished tumorigenicity of the Ad-FHIT-transduced tumor cells resulted from doomed cell viability, our results, nevertheless, indicate that the inhibitory effects on cell proliferation or cell growth demonstrated by overexpression of FHIT in established cell lines are consistent and cannot be abolished or reversed under normal physiological conditions. Moreover, the gene delivery mediated by the Ad-FHIT vector is highly efficient in vivo. Expression of Fhit could still be detected in Ad-FHIT-injected tumor cells 3 weeks after the last injection (data not shown). Furthermore, the suppression of tumor growth by exogenous FHIT overexpression is very effective, and the magnitude of tumor-suppressing activity of FHIT is comparable with that observed for p53 (24 , 25 , 35) . The transfer of exogenous wild-type p53 into cancer cells carrying mutated p53 leads to a strong induction of apoptosis and growth arrest, but is less effective in cells expressing endogenous p53. The FHIT gene, however, seems to use an alternative apoptosis pathway to perform its tumor-suppressing functions and shows an equally effective growth inhibition in both p53-deleted H1299 and wild-type p53-containing A549 lung cancer cells. Thus, gene therapy combining both p53 and FHIT tumor suppressor genes would presumably be more effective because different apoptotic pathways would be activated.
On the other hand, the results of previous experiments using tumor cell lines either transiently or stably transfected by wild-type FHIT-expressing plasmids for studying biological function of FHIT in vitro and in vivo were variable, depending on cell lines studied (14 , 15) . This suggests that some cancer cell lines may be more susceptible to physiological level of FHIT expression. However, high levels of expression in cancer cells seem to consistently induce apoptosis. Although the mechanism regulating the differential sensitivity of the FHIT-transduced cells to FHIT-mediated apoptosis is not known, the accumulation of growth-deregulating mutations may sensitize transduced cells to proapoptotic stimuli (36) .
Ad-FHIT vector has several advantages over plasmids for FHIT gene delivery and for studying the biological functions of Fhit in vitro and in vivo. First, a high efficiency of transduction (>80%) and a high level of FHIT gene expression could be easily achieved in a wide spectrum of cell types by simply adjusting the MOI of viral particles to target cells. Second, the Ad-FHIT-transduced tumor cells could be directly studied for their effect on tumorigenicity in animals without having to select for stably transduced colonies; thus, problems associated with the colony selection process and with unknown effectors or factors generated in resulting cell colonies could be avoided. Third, the Ad-FHIT vector could be directly used to evaluate the role of FHIT as a tumor suppressor gene in vivo by either i.v. or intratumoral injection of the vector into animals. Finally, the Ad-FHIT vector, by being both highly efficient at tumor suppression in vivo and reduced in toxicity and immunogenicity by virtue of E1-E3 deletion and E4 inactivation in the adenoviral vector genome (16 , 17) , could be very useful in cancer gene therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was partially supported by grants from the National Cancer Institute and the NIH, by Specialized Program of Research Excellence (SPORE) in Lung Cancer Grant P50-CA70907, by The University of Texas M. D. Anderson Cancer Center Support Core Grant CA16672, by gifts to the Division of Surgery and Anesthesiology from Tenneco and Exxon for the Core Laboratory Facility, and by an American Society of Clinical Oncology (ASCO) Young Investigator Award (to K. F.). 
2 To whom requests for reprints should be addressed, at Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, Box 109, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-6935; Fax: (713) 794-4901;
3 The abbreviations used are: FHIT, fragile histidine triad; Ad, adenovirus; HBEC, human bronchial epihelial cell; FBS, fetal bovine serum; CMV, cytomegalovirus; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MOI, multiplicity(ies) of infection; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PI, propidium iodide; FACS, fluorescence-activated cell sorting; pfu, plaque-forming unit.
Received 4/ 7/99. Accepted 5/28/99.
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