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Clinical Cancer Research Vol. 12, 3494-3501, June 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Preclinical Assessment of FHIT Gene Replacement Therapy in Human Leukemia Using a Chimeric Adenovirus, Ad5/F35

Flavia Pichiorri1,2, Francesco Trapasso1,3, Tiziana Palumbo1,4, Rami I. Aqeilan1, Alessandra Drusco1, Bradley W. Blaser1, Dimitrios Iliopoulos1, Michael A. Caligiuri1, Kay Huebner1 and Carlo M. Croce1

Authors' Affiliations: 1 Ohio State University Comprehensive Cancer Center, Columbus, Ohio; 2 Department of Biopathology and Image Diagnostics, University "Tor Vergata" of Rome, Rome, Italy; 3 Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, Catanzaro, Italy; and 4 Department of Experimental and Clinical Pharmacology, University of Catania, Catania, Italy

Requests for reprints: Flavia Pichiorri, Ohio State University Comprehensive Cancer Center, Wiseman Hall, Room 441, 410 West 12th Avenue, Columbus, OH 43210. Phone: 614-292-4354; E-mail: pichiorri.1{at}osu.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Expression of the FHIT protein is lost or reduced in most solid tumors and a significant fraction of hematopoietic malignancies. Adenovirus 5 (Ad5) virus or adeno-associated viral vectors have been used to study the tumor suppressor function of FHIT in solid tumors, but these tools have not been effective in leukemias. We have generated a chimeric FHIT-containing adenovirus composed of Ad5 and the group B adenovirus called F35 with which we have been able to efficiently infect hematopoietic cells.

Experimental Design: Infection efficiency of Ad5/F35-FHIT and Ad5/F35-GFP viruses was tested in leukemia cell lines that lacked FHIT expression, and biological effects of successful infection were assessed. An acute myelogenous leukemia, a chronic myelogenous leukemia, and four acute lymphoblastic leukemia human cell lines were examined as well as two EBV-transformed B lymphoblastoid cell lines that expressed endogenous FHIT.

Results: Two of four acute lymphoblastic leukemia cell lines, Jurkat and MV4;11, which were efficiently infected with Ad5/F35-FHIT, underwent growth suppression and massive induction of apoptosis without apparent activation of caspase-8 or caspase-2 and late activation of caspase-3. Treatment of infected cells with caspase-9 and caspase-3 inhibitors partially blocked FHIT-induced apoptosis. The two remaining infected acute lymphoblastic leukemia cell lines, Molt-3 and RS4;11, were apparently unaffected. Restoration of FHIT expression in the chronic myelogenous leukemia K562 cell line and the acute myelogenous leukemia KG1a cell line also induced apoptosis but at later time points than seen in the acute lymphoblastic leukemia Jurkat and MV4;11 cell lines. I.v. injection of Ad5/F35-FHIT-infected Jurkat cells resulted in abrogation of tumorigenicity in the NOD/SCID xenogeneic engraftment model.

Conclusion: FHIT restoration in some FHIT-deficient leukemia cells induces both antiproliferative and proapoptotic effects involving the intrinsic caspase apoptotic pathway.

Adeno-associated virus and adenovirus 5 (Ad5) infect cells through binding of the viral fiber knob to cellular coxsackie and adenovirus receptor (1, 2). These viruses are ineffective at infecting hematopoietic cells. Recently, group B adenovirus derivatives have gained attention as vectors for gene therapy because they can infect hematopoietic cells, dendritic cells, and other malignant tumor cells that are refractory to infection by commonly used adenoviruses (3, 4). Ad5 vectors incorporating F35 fiber are superior in infecting hematopoietic stem cells and dendritic cells (5, 6). Moreover, human CD46 is a cellular attachment receptor for most group B adenoviruses, including F35 (7). CD46 plays a role in protecting autologous cells from complement attack by serving as a cofactor for factor I–mediated inactivation of C3b and C4b (8, 9). Indeed, CD46 is frequently overexpressed on cancer cells, including leukemias, possibly as a mechanism to prevent complement-mediated lysis (8–12). The ability of a chimeric adenovirus to mimic the infection pattern of wild-type group B adenovirus F35, using the same CD46, could possibly improve the application of this vector for gene therapy of hematologic malignancies.

FHIT gene structure and protein expression have been examined in many types of cancers (13). FHIT expression is reduced or absent in the majority of human cancers due to genetic or epigenetic modification, although point mutations are rare. Replacement of FHIT suppresses tumorigenicity in most FHIT-deficient cancer cells thus far tested (14–17), and FHIT viral gene therapy prevents and reverses carcinogen-induced gastric cancers in Fhit-deficient mice (18, 19). Recombinant mice carrying one or two inactivated Fhit alleles are fertile and long-lived and show increased rates of spontaneous and carcinogen-induced cancers, including increased frequency of lymphomas (20, 21). From extensive FHIT overexpression studies, it is known that death of FHIT-deficient cells expressing exogenous FHIT occurs through apoptosis (18, 22–24), whereas normal and FHIT-expressing cancer cells are not affected. Transfer of the Fhit gene into Fhit-negative cancer cell lines generally results in the induction of apoptosis and suppression of the tumorigenic phenotype (16). Activation of caspase-8, caspase-9, caspase-2, and caspase-3 has been described in Fhit-induced apoptosis (17–19, 25) and Fhit reexpressing cells have been shown to be more susceptible to external apoptotic stimuli, such as serum starvation, exposure to UVC, mitomycin C, and paclitaxel, or Fas treatment compared with the parental counterpart cells (25–27), but the hierarchy of the events leading to the cell death has not been elucidated. Roz et al. (25) have reported that in NCI-H460 lung cancer cells the apoptotic mechanism induced by FHIT seems to be mediated by caspase-8 activation and is not dependent on mitochondrial mediators of apoptosis. Recently, Nishizaki et al. (24) have shown that the coexpression of FHIT and p53 synergistically inhibited tumor cell proliferation in non–small cell lung cancer cells in vitro and suppressed the growth of human tumor xenografts in nude mice by FHIT-mediated inactivation of MDM2, which thereby blocked the association of MDM2 with p53, thus stabilizing the p53 protein. Despite numerous reports on the status of the FHIT gene in multiple cancers, the biological mechanism of FHIT activity and cellular pathways associated with cell cycle and tumor suppressor function are not completely understood.

In 1997, Sugimoto et al. (28) reported that loss of FHIT function may be involved in the genesis of some human leukemias, and expression of aberrant FHIT transcripts is specific and frequent in leukemias. Iwai et al. (29) observed that FHIT expression was abolished in the majority of leukemia cases, and Hallas et al. (30) found that FHIT was lost in all T-cell acute lymphoblastic leukemia (ALL) and half of B-ALL cell lines tested. Albitar et al. (31) confirmed alteration of FHIT expression in ALL but did not find a role for FHIT as a prognostic factor. On the other hand, Wang et al. found that aberrant FHIT expression in 98 acute myelogenous leukemias (AML) correlated with a low rate of clinical remission and poor overall survival (32). Most recently, in a study of hypermethylation of the FHIT promoter in pediatric ALL (30, 33), it was found that hypermethylation of FHIT was strongly correlated with blastic morphology and hyperdiploid or a normal karyotype. Hyperdiploid B-ALLs were 23-fold more likely to exhibit FHIT promoter methylation compared with B-ALLs with TEL-AML translocations. FHIT methylation was associated with high WBC counts at diagnosis, a known poor prognostic indicator (30, 33). Recently, it has been shown that in 100% of the infant MLL cases, FHIT expression was down-regulated by DNA methylation (34). Thus, it is likely that loss of FHIT plays a role in the genesis of leukemia and that, as in epithelial malignancies, FHIT gene therapy could potentially be efficacious in hematopoietic malignancies. In this report, we test this hypothesis in leukemia-derived cells lines by using a novel Ad5/F35-FHIT chimeric virus.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The ALL (Jurkat, Molt-3, Molt-4, RS4;11, and MV4;11), AML (KG1a), and chronic myelogenous leukemia (CML; K562) cell lines were obtained from American Type Culture Collection (Manassas, VA). The MV4;11 and RS4;11 cell lines have been reported as ALL cell lines (30), although a biphenotypic B myelomonocytic leukemia origin has also been proposed (35). The EBV-transformed B lymphoblastoid cell lines were kindly provided by Dr. Bice Perussia (Thomas Jefferson University, Philadelphia, PA). All cell lines were maintained in RPMI 1640 containing 10% FCS and 0.1 mg/mL gentamicin.

Construction of chimeric adenovirus vector. In the starting Ad5/F35-FHIT, both FHIT and green fluorescent protein (GFP) cDNAs, separated by an internal ribosomal entry site, were placed under control of a cytomegalovirus promoter. The vector was linearized between the Ad5 and the Ad5' arms and then cotransfected, with the defective pAd5/F35{delta}{psi}, into 293 cells. This cotransfection allows recombination and packaging of the Ad5/F35-FHIT virus. Transfected cells were overlaid with SeaPlaque agarose, and several plaques were chosen and analyzed by Western blot for FHIT protein expression. The Ad5/F35-FHIT virus was then amplified and purified by CsCl gradient; viral particle titration was done by the TCID50 method. We also constructed a similar virus with GFP cDNA only.

Adenoviral infection. Cancer-derived cell lines were transferred to 24-well tissue culture plates (1 x 106/mL) before infection. Infection were done using 50 to 100 multiplicities of infection (MOI) per target cell after thawing titrated virus stocks at 37°C, mixing appropriate volume of virus concentrate with RPMI-5% FCS, and adding the mixture to target cells. After 12-hour incubation at 37°C, cells were washed in warm PBS and resuspended in fresh medium supplemented with 10% serum. Infected cells were examined daily by inverted light and fluorescence microscopy.

Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell counting. At 48, 72, and 96 hours postinfection, cells were collected, washed with PBS, and resuspended in cold 70% ethanol. For analysis, cells were spun down, washed in PBS, and suspended in 0.1 mg/mL propidium iodide/Triton X-100 staining solution (0.1% Triton X-100, 20 mg/mL, 0.2 mg/mL DNase-free RNase A) for 30 minutes at room temperature and analyzed by flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done with a kit (Sigma Chemical Co., St. Louis, MO) as recommended by the manufacturer. For cell growth kinetics, 1 x 105 cells per well were cultured in 96-well culture plates. The number of cells per well was counted at indicated times in triplicate, excluding the dead cells by trypan blue staining.

Caspase inhibition assay. The caspase inhibitors zVADfmk (general), zDEVDfmk (caspase-3 and caspase-7), zIETDfmk (caspase-8), and zLEHDfmk (caspase-9) were purchased from BD Biosciences (San Jose, CA). Jurkat cells were seeded (1 x 105 per well) in 96-well culture plates and the infection was done using 100 MOI. The cells were incubated at 24, 48, 72, and 96 hours with different concentrations of caspase inhibitors (25-80 µmol/L) and the media with the inhibitors were replaced daily. MTT assay was done with a kit as recommended by the manufacturer.

Immunoblot analysis. Immunoblot analysis was done by standard protocols (36). Briefly, 5 x 105 cells were cultured in 60-mm-diameter dishes and lysed on ice in 300 µL lysis buffer. Protein lysates were fractionated on 4% to 20% linear gradient SDS-PAGE gels and electroblotted to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were blocked with 5% skim milk and incubated with the indicated antisera. After reaction with an appropriate secondary antiserum, the immunoreactive bands were detected by the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's protocol. The following antisera and dilutions were used: rabbit polyclonal anti-FHIT at 1:1,000 (Zymed, South San Francisco, CA), mouse monoclonal anti-caspase-2 at 1:1,000 (BD Biosciences), mouse monoclonal anti-caspase-3 at 1:500 (BD PharMingen, San Diego, CA), rabbit polyclonal anti-caspase-8 at 1:250 (Chemicon, Temecula, CA), rabbit polyclonal anti-caspase-9 at 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-GADPH at 1:3,000 (Sigma).

Immunocytochemistry. Cells were washed in PBS and fixed in 4% PBS-buffered formalin for 10 minutes before drying on glass slides. After blocking with normal donkey serum, slides were incubated with rabbit anti-human FHIT serum (Zymed; diluted 1:500 in PBS with 1% bovine serum albumin) overnight at 4°C followed by biotinylated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 hour at room temperature. Slides were then incubated for 30 minutes with ABC-streptavidin and detection was done using 3,3'-diaminobenzidine following the manufacturer's instruction (Santa Cruz Biotechnology staining kit).

In vivo tumorigenicity. Animal studies were done according to institutional guidelines. Jurkat cells were seeded in six-well dishes and either in vitro mock infected or infected with Ad5/F35-FHIT or Ad5/F35-GFP at a MOI 100. Twenty-four hours after infection, 2 x 107 viable cells were resuspended in 500 µL PBS and injected by tail vein into each of 5 to 10 female NOD/SCID mice (Charles River, Cambridge, MA). At 10 days postinjection, mice were sacrificed and engraftment was assessed by flow cytometric detection of human CD45+ Jurkat cells. Anti-human CD45 was purchased from BD Pharmacon (San Jose, CA).

Generation of K562/TP53 stable transfectant clones. Clones of K562 cells expressing human p53 were generated by transfection of K562 p53-null cells with pCMV-p53 (BD Biosciences) using the nucleofection protocol (Amaxa Biosystem, Gaithersburg, MD). p53-expressing clones were selected by limiting dilution in 1.0 mg/mL G418 (Invitrogen, Carlsbad, CA) and were screened for p53 expression by PCR using the following primers: forward 5'-GGAATTCCACGACGGTGACAC-3' and reverse 5'-GGAATTCCGAAAAGTGTTTCT-3'.

Statistical analysis. Data obtained from in vivo and in vitro experiments 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. P < 0.05 was considered significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In vitro infection of leukemia cells with adenoviral FHIT. The leukemia cell lines were first assessed for endogenous FHIT expression by immunoblot analysis. Endogenous FHIT was undetectable in Molt-3, RS4;11, MV4;11, K562, KG1a cells; after prolonged exposure, weak FHIT expression was detected in Molt-4 and Jurkat cells (Fig. 1A ) compared with the FHIT-positive EBV-transformed B large-cell lymphomas. We then tested the infection efficiency of Ad5/F35-FHIT virus in the five ALL-derived cell lines (Jurkat, Molt-3, Molt-4, RS4;11, and MV4;11), one AML-derived cell line (KG1a), one CML-derived cell line (K562), and two FHIT-positive EBV-positive large-cell lymphomas. Efficient infection was confirmed by GFP expression analysis using fluorescence microscopy (Table 1 ), whereas FHIT expression in the leukemia cell lines was analyzed by Western blot (Fig. 1B) and immunocytochemistry (Fig. 1C). In Fig. 1B, the slower migratory FHIT band in these hematopoietic cells does not represent phosphorylated FHIT (30, 34) but another modification that we are currently investigating by mass spectrometry. As shown in Fig. 1C, FHIT expression was detected exclusively in the cytoplasm.


Figure 1
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  		Fig. 1. A, expression of endogenous FHIT in leukemia cell lines. Five ALL-derived cell lines (Jurkat, Molt-3, Molt-4, RS4;11, and MV4;11), the AML-derived cell line KG1a, the CML-derived cell line K562, and the EBV-transformed B large-cell lymphomas were examined by Western blot. A high level of endogenous FHIT was found only in the EBV large-cell lymphomas. B, FHIT expression in Ad5/F35-FHIT-infected cell lines by Western blot. C, immunocytochemistry analysis. FHIT expression and cellular localization in leukemia cell lines at 48 hours after Ad5/F35-FHIT infection. FHIT was abundantly expressed in each infected cell line tested, localized primarily in the cytoplasm.

 

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  		Table 1. Characteristics of leukemia-derived cell lines

 
Analysis of cell growth in leukemia cell lines infected with Ad5/F35-FHIT. Leukemia cell enumeration following Ad5/F35-FHIT and Ad5/F35-GFP infection showed dramatic inhibition in the Ad5/F35-FHIT group for Jurkat (T-ALL), MV4;11 (B-ALL), K562 (CML), and KG1a (AML) cells when compared with Ad5/F35-GFP-infected cells, whereas proliferation in RS4;11 (B-ALL) and Molt-3 (T-ALL) was unaffected despite equally efficient infection (Supplementary Fig. S1). For cell growth kinetics, the number of cells was counted at indicated times in triplicate, excluding the dead cells, by trypan blue staining.

Analysis of apoptosis and cell cycle kinetics in Ad5/F35-FHIT-infected leukemia cell lines. A large percentage of the two ALL cell lines, Jurkat and MV4;11, were in the apoptotic/necrotic fraction by 72 to 96 hours after Ad5/F35-FHIT infection, 61.2% and 60.4%, compared with cells infected with Ad5/F35-GFP, 12.2% and 30.6% respectively. In contrast, only a modest fraction of FHIT-infected K562 and KG1a cells underwent apoptosis (27% for K562 and 21.5% for KG1a) and did so at a slower rate, whereas the FHIT-infected B-ALL cell line RS4;11 and the T-ALL cell line, Molt-3 did not undergo significant apoptosis (3.58% for RS4;11 and 11.9% for Molt-3; Fig. 2 ). Molt-3 T-ALL-derived cells showed cell cycle arrest in G2-M (16.4%) at 96 hours compared with GFP-infected (7.67%) and mock-infected (8.25%) cells but no apoptosis. Moreover, we studied cell growth of this ALL cell line for 7 days after FHIT transduction and did not observe significant cell growth inhibition (Supplementary Fig. S1). Mock- or Ad5/F35-GFP-infected cell lines did not show changes in cell cycle profile. The lack of apoptosis in several Ad5/F35-FHIT-infected cell lines excludes a general cytotoxic effect of FHIT overexpression in cell lines lacking endogenous FHIT. Indeed, the heterogeneity of responses to Ad5/F35-FHIT infection by these cell lines is similar to results of an earlier study of human esophageal cancer cell lines in which four of seven cell lines underwent apoptosis or cell cycle arrest after FHIT overexpression (16).


Figure 2
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  		Fig. 2. FACScan analysis of cell cycle distribution and apoptosis. Distribution of infected cells in the cell cycle was analyzed by propidium iodide (PI) staining at 48, 72, and 96 hours after infection. Representative experiments at 96 hours. The sub-G1 population was ~60% at 96 hours postinfection in Jurkat and MV4;11 cell lines. Molt-3 and RS4;11 did not show differential effects of FHIT and GFP transduction.

 
Analysis of proapoptotic signaling in FHIT-expressing cells. To investigate molecular mechanisms involved in oncosuppressive activity of Ad5/F35-FHIT, we studied the expression of proteins involved in the apoptotic cascade: caspase-3, caspase-8, caspase-2, and caspase-9. In Jurkat, MV4;11, and K562 cell lines, we observed reduced levels of pro-caspase-3 at 96 hours after Ad5/F35-FHIT infection compared with controls, suggesting activation of caspase-3. Caspase-8 and caspase-2 were not reduced (Fig. 3A ) and pro-caspase-9 was barely detectable. The pro-caspase-3 level was not altered in RS4;11 or Molt-3 cells that did not undergo apoptosis after Ad5/F35-FHIT infection (Fig. 3A). To better understand which caspase pathway was involved in the late stage of FHIT-induced cell death, we evaluated in Jurkat cells the protective effect of general (zVADfmk) and respective specific caspase-3, caspase-9, and caspase-8 inhibitors, zDEVDfmk, zLEHDfmk, and zIETDfmk (Fig. 3B). We found that at 72 hours caspase-9 inhibition caused a partial block of FHIT-induced apoptosis (P = 0.001), whereas at 96 hours caspase-9 (P = 0.006), caspase-3 (P = 0.047), and pan-caspase (P = 0.019) inhibitors blocked FHIT-induced apoptosis. Each experimental point was repeated in quadruplicate in two independent experiments. Moreover, we studied p53 status and induction of apoptosis in Ad5/F35-FHIT-infected cells but did not observe any correlation (Table 1). To investigate this question further, we generated K562 cells stably expressing a low level of wild-type p53 (Fig. 4A ). We did MTT growth tests on four different p53-positive colonies compared with parental K562 and three colonies transfected with the empty vector. Stable transfection of wild-type p53 into the K562 cell line did not alter the fraction or rate of apoptosis or colony formation following Ad5/F35-FHIT infection (Fig. 4B).


Figure 3
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  		Fig. 3. A, modulation of apoptosis-related proteins in Ad5/F35-FHIT-infected leukemia cell lines. Overexpression of FHIT in Jurkat, MV4;11, and K562 cells induces cleavage of pro-caspase-3 at 96 hours postinfection, shown as reduced intensity of the 35-kDa band. In Jurkat and MV4;11 cells, a 3-fold reduction of caspase-3 was observed, but only a small difference in K562 cells. No difference in caspase-3 protein was detected in Molt-3 and RS4;11 cells. No difference, in any cell line analyzed, was detected in caspase-8 or caspase-2 at 96 hours postinfection. Exogenous FHIT expression is shown in Ad5/F35-FHIT-infected leukemia cells. B, kinetics of caspase inhibition assay in Jurkat cells by MTT assay. Effects of the general caspase inhibitor zVADfmk and inhibitors of caspase-3 and caspase-7 (zDEVDfmk), caspase-8 (zIETDfmk), and caspase-9 (zLEHDfmk) on FHIT-transduced and untransduced Jurkat cells at 72 and 96 hours postinfection. Each inhibitor was used at a final concentration of 25 to 80 µmol/L. Columns, mean in quadruplicate; bars, SD. P < 0.05 was considered as statistically significant. At 72 hours, caspase-9 inhibitor partially blocked FHIT-induced apoptosis (P = 0.001), whereas at 96 hours caspase-9 (P = 0.006), caspase-3 (P = 0.047), and the pan-caspase inhibitor (P = 0.019) blocked FHIT-induced apoptosis.

 

Figure 4
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  		Fig. 4. A, expression of TP53 mRNA in K562 TP53 stable clones by reverse transcription-PCR. Reverse transcription-PCR products in K562 empty vector colonies (lanes 2 and 3), K562 p53-expressing stable colonies (lanes 4-10), and the parental cell line (lane 1). B, MTT cell growth assay of K562 p53 stable colony versus K562 empty vector stable colony and K562 parental cells (infected with Ad5/F35-FHIT at MOI 50). Columns, average of four independent assays; bars, SD. Cells (5 x 105) were seeded in 96-well plate and MTT assay done as recommended by the manufacturer at 24, 48, 72, and 96 hours. No significant difference in cell viability was observed (P > 0.05) after Ad5/F35-FHIT infection of stable p53-expressing clones. The kinetics of Fhit-induced loss of cell viability seems unaffected by the presence of p53 expression.

 
Tumorigenicity of Ad5/F35-FHIT-infected Jurkat cells in vivo. We tested different cell lines (K562 and MV4;11) to establish an engraftment model and found that the Jurkat T-ALL cell line provided the best results. We examined the tumorigenic potential of chimeric Ad5/F35-FHIT-infected Jurkat cells in NOD/SCID mice compared with GFP- and mock-infected cells (Fig. 5 ). We injected transduced Jurkat cells 24 hours after infection, when cells were still healthy and viable. The flow cytometric time course of transduced Jurkat cells at 24, 48, 72, and 96 hours is shown (Fig. 5A). FHIT protein was expressed at the same time points only in Ad5/F35-FHIT-infected cells as expected (Fig. 5B).


Figure 5
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  		Fig. 5. A, flow cytometric time course of FHIT-, GFP-, and Mock-infected Jurkat cells at 24, 48, 72, and 96 hours. The cells were i.v. injected 24 hours after infection when the FHIT-transduced cells did not show a significant subdiploid fraction. B, FHIT protein was expressed at the same time points only in Ad5/F35-FHIT-infected cells. C, flow cytometric analysis of engrafted Jurkat cells. Representative results of flow cytometric analysis of the WBC of recipient mice. NOD/SCID mice injected with 20 x 106 Jurkat cells preinfected with Ad5/F35-FHIT did not show CD45+ human cells (0.2%) in the blood 10 days after injection. The mice previously injected with wild-type uninfected cells or Ad5/F35-GFP-infected cells show 25% and 12% engraftment of Jurkat cells in the mouse blood, respectively. D, incidences of tumor engraftment presented as average percentage of WBC for each group of five to seven mice (mean ± SD).

 
The mice WBC were monitored by cell counts and by flow cytometric analysis for human CD45+ cells. Ten days after injection of Ad5/F35-FHIT-infected Jurkat cells, we did not find any evidence of engraftment in blood, whereas the mice injected with the Ad5/F35-GFP-infected Jurkat cell lines had 12% of human CD45+ cells in the blood of 47% of mice and the mice injected with the mock-infected Jurkat cell lines had 25% of human CD45+ cells in the blood of 83% of mice (Fig. 5C and D). After sacrifice, we also detected a small percentage of engrafted cells in the spleens of mice injected with Ad5/F35-GFP- and mock-infected Jurkat cells but not in the spleens of mice injected with Ad5/F35-FHIT-infected Jurkat cells.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Loss of FHIT expression in human leukemias is well documented (28–34), but to date an effective gene replacement approach has not been studied in any preclinical model. This report marks the first study to our knowledge that examines of the effectiveness of a FHIT reexpression strategy in hematologic malignancies.

We first evaluated the effect of FHIT viral gene transfer into human leukemia-derived cancer cell lines in vitro. We observed growth suppression and massive induction of apoptosis in two of four ALL-derived cell lines, Jurkat and MV4;11, despite the absence of endogenous FHIT in all four cell lines. Restoration of FHIT expression in K562 (CML) and KG1a (AML) cell lines also caused cell death but only after a prolonged period compared with the two FHIT-sensitive ALL cell lines. In epithelial tumors, the apoptotic effect of FHIT was also relatively delayed after infection.

The mechanism of tumor suppression by FHIT is not fully understood. Several studies have shown that FHIT overexpression leads to activation of one or both main proapoptotic caspase cascades, mitochondrial-mediated caspase-3 activation through caspase-9 (intrinsic/mitochondrial pathway) or caspase-3 activation by caspase-8 (extrinsic/cell death receptors pathway; refs. 17–19, 25, 26, 37). Sevignani et al. (17) described activation of caspase-2 after Ad-FHIT infection of FHIT-negative breast and colon cancer cell lines, whereas activation of caspase-8 and caspase-9 was not a common event. In the present study, we have found activation of caspase-3 at 96 hours after chimeric Ad5/F35-FHIT infection, when the frequency of cell death was >60%, but we did not observe activated caspase-8 or caspase-2 at 96 hours in Jurkat or MV4;11 cells. In Jurkat cells, we detected a protective effect at 72 hours of caspase-9 inhibitor and at 96 hours of caspase-9, caspase-3, and general caspase inhibitors. Our data suggest that the intrinsic mitochondrial pathway is involved in the late stages of FHIT-induced apoptosis. Previous data on apoptosis triggered by the FHIT protein in epithelial tumors showed involvement of both cytoplasmic and mitochondrial pathways (18, 38). In this study, we hypothesized that in leukemia cell lines the mitochondrial pathway is primarily involved in the late stage of FHIT apoptotic process.

Regarding FHIT and TP53 status, five of the endogenous FHIT-negative cancer cells, K562, KG1a, Jurkat, MV4;11, and RS4;11, lack wild-type p53 expression and the Molt-3 cell line expresses wild-type p53 (39). There was no correlation between p53 expression and FHIT apoptotic activity. Analysis of stable human TP53-positive K562 colonies confirmed these results; p53 reexpression did not affect the ability of FHIT to induce apoptosis, confirming that the apoptotic effect of FHIT is independent of wild-type p53 expression in these leukemia cells. Thus, the action of FHIT in association with p53 in hematopoietic malignancies is different from that reported for non–small cell lung cancer cells, where FHIT and p53 were reported to have a synergistic effect in apoptosis (24).

To our knowledge, this is the first study to show evidence of suppression of in vivo tumorigenicity of leukemia cells by a chimeric adenovirus transducing the FHIT gene. The work represents a technical advance because the chimeric virus was able to transduce Jurkat cells with high efficiency without a nonspecific toxic effect. In our in vivo model, we injected transduced Jurkat cells 24 hours after infection, when cells had only modest evidence of FHIT expression and showed no signs of apoptosis. Engraftment of Jurkat cells reexpressing the FHIT gene was completely suppressed in all injected mice.

Taken together, these results show that the Ad5/F35-FHIT virus that infects via the CD46 receptor may be a valuable tool for short-term expression of the transgene in cancers that express abundant CD46 and may ultimately find clinical applications in gene therapy of hematologic malignancies, likely in combination with autologous bone marrow transplantation. Discrimination between high and low CD46 receptor density provides a compelling basis for the infection efficiency of Ad5/F35 virus and bears directly on the application of these vectors to more efficiently target tumor tissues than healthy tissues (8–12). Moreover, it may be that FHIT expression engages a late intrinsic apoptotic response to repress some but not all forms of leukemia. Clarification of this mechanism may suggest novel clues to FHIT function in future studies.


   Acknowledgments
 
We thank Drs. Rodolfo Iuliano and George Calin for advice and suggestions and Prof. Luigi Giusto Spagnoli (Director of the Department of Biopathology and Image Diagnostics, University "Tor Vergata" of Rome) for his scientific support.


   Footnotes
 
Grant support: Kimmel Scholar Award (R.I. Aqeilan) and National Cancer Institute/NIH, USPHS grants CA77738, CA78890, CA89341, and CA56036.

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: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 11/28/05; revised 3/ 4/06; accepted 3/24/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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