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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 3, 2003 as doi:10.1096/fj.03-0241fje. |
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* Kimmel Cancer Center and
CNS Gene Therapy Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
3Correspondence: Kimmel Cancer Center, BLSB, Room 1050, 233 S. 10th St., Philadelphia, PA 19107, USA. E-mail:C_Croce{at}lac.jci.tju.edu
SPECIFIC AIMS
Fhit expression is reduced in most human cancers and mice carrying one or two inactivated Fhit alleles are hypersensitive to N-nitrosomethylbenzylamine (NMBA) -induced forestomach tumors. Our aim was to investigate the kinetics and mechanism of tumor reversal by oral delivery of FHIT expression viruses.
PRINCIPAL FINDINGS
1. Reversal of upper gastric cancers by FHIT gene delivery
More than 200 Fhit +/- mice were treated with the carcinogen NMBA, using a regimen that would induce forestomach and squamo-columnar junction (SCJ) tumors in all Fhit +/- mice; mice were then divided into groups to be treated with viruses at various times post-MBA treatment. The viruses were adenoviral (Ad) or adeno-associated (AAV) viruses carrying genes for GFP, FHIT, or the hydrolase "dead" FHIT mutant H96N, with a glutamine at codon 96 to replace the wild-type histidine codon. The control Ad-GFP and AAV-GFP viruses were delivered to mice 2 days post-MBA, and wild-type and mutant FHIT viruses were delivered at 1, 2, 21, or 42 days post-NMBA treatment. Mice were killed at various times after treatment, and stomachs were removed and assessed for tumors (Fig. 1
). Control NMBA-treated mice, receiving no viral therapy were assessed at days 2, 10, 37, 49, 58, 84, and 140 post-NMBA to delineate the time course of tumor development. Multiple small tumors were apparent at 37 days in 2 of 3 mice and by 49 days all mice showed an average of 7 large (>1 mm) tumors per forestomach. By 84 days, 18 of 18 mice showed >10 large or fused forestomach tumors (Fig. 1a
); by 140 days most of these control mice exhibited invasive carcinomas of the forestomach. All mice receiving GFP expression viruses also developed 
10 large or fused tumors per forestomach by 84 days post-NMBA (Fig. 1b
). In contrast, as illustrated in Fig. 1c-e
, there was a dramatic reduction in tumor incidence, size, and multiplicity in the forestomachs of mice receiving the FHIT gene via a single oral dose of FHIT virus. Control animals killed 2 to 10 days post-NMBA basically did not exhibit tumors; thus, viral gene delivery administered on day 1 or 2, which caused highly significant reductions in tumor multiplicity and size at end point, very likely involved prevention of development of tumors through Fhit-induced apoptosis of preneoplastic epithelial cells (see below). On the other hand, control animals had each developed 7 large tumors by 49 days post-NMBA, so that animals infected at day 42 post-NMBA and showing 3, mostly small, tumors per mouse at end point must have had therapeutic regression or elimination of most of their tumors. As for gene therapy of forestomach tumors with the Fhit mutant protein, the AAV-FHITH96N-treated animals showed a highly significant reduction in tumor multiplicity to 2 per forestomach at end point; the Ad-FHITH96N-treated animals showed a trend toward reduction in tumor multiplicity at 84 days that did not reach significance. The group of animals treated twice with Ad-FHIT (at 2 and 21 days post-NMBA) did not show significant reduction in tumor incidence or multiplicity.
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Histological examination of mouse forestomach sections showed an array of lesions, including hyperplasia, focal hyperplastic lesions, papillomas, and carcinomas. For The AAV-FHIT group killed at 84 days there were very few carcinomas (3/47), a significantly (P=0.05) reduced number relative to the number of mice with carcinomas (17/50) in the control groups (control, AAV-GFP and Ad-GFP). The frequency of carcinomas in the Ad-FHIT-treated group was lower than the control group but the difference was not significant. To determine whether tumor reduction was long lasting, we compared a group of mice treated with Ad-FHIT and observed for 140 days with control NMBA-treated mice at 140 days. At this point, 9/9 control mice showed 14 large or fused tumors per forestomach, while the Ad-FHIT-treated mice showed only 4–6 tumors per forestomach, with 5 mice showing only small tumors (P<0.001).
2. Cell proliferation in forestomach epithelia
Histopathology and PCNA staining of epithelia for identification of proliferative cells showed that the epithelial layers of FHIT virus-treated mice were thin, composed of 5–10 cell layers, with PCNA staining mainly in the basal layer. The forestomach carcinomas observed in control and GFP virus-treated animals showed intense PCNA staining, indicating proliferation of cells throughout the tumor. Histopathology and PCNA staining also illustrated the point that tumors regressed within 1 or 2 wk after virus treatment, as shown by thin epithelia and basal layer PCNA staining in sections from mice killed within 7–14 days after infection.
3. Apoptosis
TUNEL analysis and immunohistochemical assessment of proapoptotic Bax and antiapoptotic Bcl-2 proteins were used to determine whether Fhit-induced tumor suppression involved apoptosis. AAV-GFP mice that received virus on day 2 and were killed on day 10 showed infrequent TUNEL-positive nuclei, mostly in the outermost cell layer (Fig. 2
a). Near adjacent sections displayed weak, diffuse expression of Bax and overexpression of Bcl-2 in basal and focal hyperplastic areas (Fig. 2a
). Ad-GFP control mice that received virus on day 2 and were killed on day 84 showed occasional TUNEL-positive nuclei in the proliferative forestomach epithelium and invasive squamous cell carcinomas were observed in some sections (Fig. 2b
). Accordingly, adjacent forestomach sections showed very weak Bax but strong Bcl-2 expression in these lesions (Fig. 2b
). These data demonstrate that neither NMBA nor viral vectors induced apoptosis in forestomach epithelium. In contrast, mice that received FHIT virus on days 2, 21, and 42 and were killed 7–14 days postinfection or at 84 post-NMBA often displayed clusters of TUNEL-positive nuclei in areas of restored or still proliferative epithelium. Overexpression of the proapoptotic Bax protein and weak staining of the antiapoptotic Bcl-2 protein were often found in the areas of increased apoptotic activity. For example, an AAV-FHIT mouse that received viral FHIT gene on day 21 and was killed on day 35 displayed >10 clusters of TUNEL-positive nuclei in a forestomach section (a cluster is shown in Fig. 2a
). Near adjacent sections exhibited overexpression of Bax in a corresponding area of apoptotic activity with Bcl-2 staining in the basal cell layer only (Fig. 2a
). Similarly, a mouse that received AAV-FHIT on day 2 and was killed on day 84 showed many TUNEL-positive nuclei in a restored epithelium with strong Bax expression and weak Bcl-2 staining (Fig. 2b
). An Ad-FHIT-treated mouse (infected day 21 and killed day 84) showed several TUNEL-positive nuclei in a proliferative area of the forestomach (Fig. 2b
). Near adjacent sections showed two stripes of strong Bax staining that covered all cell layers and Bcl-2 staining in basal and suprabasal cell layers of the still proliferative epithelium (Fig. 2b
). Thus, Bax expression and apoptosis were induced and sustained after the application of FHIT gene therapy, thereby contributing to inhibition and reversal of tumorigenesis.
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CONCLUSIONS
A pilot study suggested that viral FHIT delivery could reduce tumor burden by intervention before tumors developed. In the current FHIT replacement study, we wanted to determine whether established tumors would be eradicated when FHIT was delivered late after carcinogen. We have examined the effect of FHIT delivery on tumor incidence, multiplicity, and size in parallel with assessment of expression of proliferative and apoptotic proteins.
When AAV-FHIT or Ad-FHIT virus was delivered 21 or 42 days post-NMBA, when early or even large tumors were already established, the tumor incidence at end point for both viruses was significantly reduced, except for the animals treated with a second dose of Ad-FHIT. In fact, these animals showed the highest multiplicity of any FHIT treatment group. It is likely that an immune response to the second dose contributed to the poor outcome, leading us to suggest that treatment of these animals with a second dose of AAV-FHIT might have produced an optimal outcome. Overall, AAV-FHIT virus gave the best therapeutic and preventive effect in this cancer model. We know from studies of in vitro Ad-FHIT and AAV-FHIT infection of human tumor cells that Fhit overexpression induces apoptosis in susceptible cancer cells but not in normal or some cancer cell lines. Thus, we assessed expression of Bax and Bcl-2 proteins and frequency of apoptotic cells in sections of forestomach tissues. The viral GFP-infected forestomachs showed opposite Bax and Bcl-2 staining patterns compared with viral FHIT infected forestomach sections, reflecting the ongoing apoptosis in the nonproliferating epithelia of Fhit expressing sections, with concomitant overexpression of the apoptotic protein, Bax. We believe that the dramatic reduction in already established forestomach tumors provides a strong basis for planning of clinical trials for therapy of specific cancer targets.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0241fje; doi: 10.1096/fj.03-0241fje 
2 These authors contributed equally.
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