WO2002064171A1 - Adenoviral transduction of fragile histidine triad (fhit) into cancer cells - Google Patents

Adenoviral transduction of fragile histidine triad (fhit) into cancer cells Download PDF

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WO2002064171A1
WO2002064171A1 PCT/US2002/004154 US0204154W WO02064171A1 WO 2002064171 A1 WO2002064171 A1 WO 2002064171A1 US 0204154 W US0204154 W US 0204154W WO 02064171 A1 WO02064171 A1 WO 02064171A1
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fhit
cells
cancer
adenoviral
gene
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Carlo M. Croce
Frances Kay Huebner
Kristoffel R. Dumon
Hideshi Ishii
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Thomas Jefferson University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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Definitions

  • the invention was made in part with government support under grants CA77738, CA51083, CA21124, and CA56336 awarded by the National Cancer Institute. The government has certain rights to the invention.
  • This invention relates to the fields of molecular biology and-gene therapy and is directed to a method of treating cancer, and, more particularly, to inducing apoptosis in esophageal cancer cells by gene delivery using viral vectors expressing the Fragile Histidine Triad ("FHIT') gene.
  • FHIT' Fragile Histidine Triad
  • Human chromosome 3p is one of the chromosomal regions most frequently deleted in human tumors, including those of lung, breast, esophagus, and bladder. (1). Positional cloning of this region has led to identification of the FHIT gene, which encodes a member of the histidine triad protein superfamily. (2-5). The FHIT gene encompasses the FRA3B fragile site (2, 6), and a genomic locus which is frequently involved in allelic loss, genomic rearrangement, and cytogenetic abnormalities in tumors (2-4).
  • FHIT locus Numerous studies have shown genomic alterations at the FHIT locus, such as biallelic deletions, translocations, and the loss of Fhit protein expression in many human cancers (2-4), including those of the esophagus, lung, breast, cervix, and bladder. While point mutations within the FHIT gene are rare (7-9), deletions are extremely common (2-4), and, less frequently, methylation is involved in Fhit inactivation (10). Nucleotide sequence analysis of the FHIT locus in tumor cell lines indicates that long interspersed nuclear element ("LINE") and long terminal repeal (“LTR”) sequences are involved in homologous recombinations at the deletion endpoints in most cancers. (11, 12). Presumably because FHIT encompasses the fragile region, which is carcinogen sensitive, the FHIT gene is susceptible to damage caused by environmental carcinogens, leading to clonal expansion of Fhit negative cells.
  • LINE nuclear element
  • LTR long terminal repeal
  • Esophageal cancer one of the most deadly human tumors, occurs worldwide, and its incidence is increasing in the Western world. (13, 14).
  • Therapeutic approaches for esophageal cancer include not only conventional therapies, such as surgical removal and radiation treatment, but gene therapy strategies, such as introduction of the tumor suppressor pl6/LNK4 (15); the expression of LL2, IL6 and GM-CSF gene products (16, 17); and the transduction of the herpes simplex virus- thymidine kinase gene (18, 19).
  • Previous FHIT gene replacement experiments mainly involved stable transfectants of endogenous Fhit-negative tumor cells to assess the biological function of Fhit protein.
  • Stable exogenous Fhit expression in Fhit-negative lung, gastric, and renal cancer cells resulted in inhibition of tumor cell growth (22-24), at least in part due to induction of apoptosis (23).
  • Ji et al. demonstrated that reintroduction of Fhit protein by adenoviral-EfflT gene transduction in lung and head- ' and-neck cancer cell lines caused apoptosis and inhibition of tumorigenicity. (25).
  • Other studies have questioned the status of FHIT as a tumor suppressor, based on observations of tumorigenicity of stable FHIT transfectants. (26, 27).
  • the present invention provides evidence that transient Fhit expression after adenoviral-EH/r transduction of esophageal cancer cells suppresses cancer cell growth in vitro. Additional evidence demonstrates that tumorigenicity is abrogated by adenoviral FHIT infection in esophageal cancer cell lines.
  • the present invention is directed to a method of inhibiting the growth of cancer cells and tumor development through adenoviral transduction of FHIT.
  • SABE means "small air way bronchial epithelial"
  • Z-NAD-fmk means "benzyloxycarbonyl-valinyl-alaninyl-aspartyl(O- methyl)-fluoromethylketone
  • GFP means "green fluorescent protein
  • MTS means "[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium inner salt]"
  • cancer is any disorder of the class of diseases of animals characterized by uncontrolled cellular growth, which disesase can be either premalignant or malignant.
  • a nucleic acid encoding a FHIT gene, or derivative thereof is delivered to the cancer cells.
  • a Fhit protein, or derivative thereof is expressed by the FHIT gene, or derivative thereof, in the mammalian cells, thereby inducing apoptosis.
  • the nucleic acid encoding the EHH gene, or derivative thereof is in a vector.
  • the vector is a viral vector.
  • the viral vector is a recombinant adenovirus.
  • the mammalian cancer cells are esophageal cancer cells.
  • Figure 1 Adenoviral-EHZT expression in esophageal cancer cell lines.
  • Adenoviral-EHiT (lanes 1-7) and adenoviral-EHZT-GEP (lanes 8-14) vectors are used for infection of esophageal cancer T ⁇ 1 (lanes 1 and 8), T ⁇ 2 (lanes 2 and 9), TE4 (lanes 3 and 10), TE10 (lanes 4 and 11), TE12 (lanes
  • Figure 2 Flow cytometry of infected esophageal cancer cell lines.
  • Figure 3 Increased apoptosis of adenoviral-EHiT infected esophageal cancer cell lines.
  • Figure 4 Increased apoptosis of adeno viral -FHIT infected HeLa cells.
  • the cervical cancer cell line, HeLa is infected with viral vectors.
  • adenoviral-GEP (lanes 1-8, 10 and 12) or adenoviral-GEP (lanes 9, 11 and 13) at MOI 20 for 72 hours.
  • Cell lysates 50 ⁇ g protein
  • Immunoblot analysis is performed with PARP, Bid, caspase 9, and ⁇ -actin antibodies as indicated.
  • Precursor forms of PARP, Bid, and procaspase 9 are cleaved in T ⁇ 4 and TE14 cells.
  • Figure 7 In vitro cell growth of esophageal cancer cell lines after adenoviral-EHH expression.
  • A, B MTS assay of T ⁇ 14 (A) and TE4 (B) cells infected with adenoviral-EH/P (•), adenoviral-EH/E-GEP ( ⁇ ), adenoviral-GEP (O) and adenoviral- A CZ (D) vectors at MOIs 30, 10, 5, and 2 for the indicated periods. Culture medium is renewed daily. Data on infection of T ⁇ 4 cells at MOI 30 are shown. Results are shown as an inhibition (%) by comparison with non-infected control experiments. Each data point shown is an average of four independent assays. (C) Growth curve for TE12 cells.
  • the cell number is counted by trypan blue exclusion at the indicated times after infection with adenoviral-EH/P (•), adenoviral-EH/P-GEP ( ⁇ ), adenoviral-GEP (O), and adenoviral- ACZ (D) vectors.
  • TE14 cells are infected by adenoviral-EH/P (•), adenoviral-EH/P-GEP ( ⁇ ), adenoviral-GEP (O), and adenoviral-LACZ vector
  • C Immunohistochemical analysis of excised tumor tissues. The tissues are stained with anti-human Fhit antibody.
  • tumor infected with adenoviral- FHIT upper left; magnification, x400
  • II tumor infected with adenoviral- LACZ (upper right; magnification, x250)
  • III tumor infected with adenoviral- GFP (lower left; magnification, x250)
  • IV tumor infected with adenoviral-EHTP-GEP (lower right; magnification, x400).
  • the FHIT gene has been identified in a region at chromosome 3pl4.2 that is deleted in many tumors, including esophageal cancer. Genetic alterations of the FHIT gene are frequently observed in many human cancers, and a tumor suppressor function of Fhit has been demonstrated. In the present invention, the biological effects of adenoviral-EH/P transduction in esophageal cancer cell lines is examined.
  • the cervical cancer cell line, ⁇ eLa, and esophageal cancer cells, T ⁇ 1, T ⁇ 2, TE4, TE10, TE12, TE13, and TE14 are maintained in RPMI 1640 medium with 10% fetal bovine serum.
  • Cells can be those of any animal, including, but not limited to, animals such as cows, pigs, horses, etc., and are preferably those of a mammal, and most preferably human.
  • SABE cells are obtained from Clonetics, Walkersville, MD, and cultured as recommended.
  • caspase inhibitors are purchased from Calbiochem (San Diego, CA): Z-VAD-fmk, an inhibitor for caspases 1, 3, 4 and 7; Z-DEND-fmk, an inhibitor for caspases 3, 6, 7, 8 and 10; and Z-IETD-fmk, an inhibitor for caspase 8.
  • cD ⁇ As for Gfp and lacZ are obtained from expression vectors (Clontech, Palo Alto, CA). Each cD ⁇ A is ligated into an adenoviral backbone D ⁇ A (Quantum, Montreal, Canada).
  • adenoviral vectors an adenoviral-EH/P-GEP vector that encodes two separate proteins through the internal ribosome entry site, an adenoviral-EH/P vector, an adenoviral-GEP vector, and an adenoviral-LACZ vector are constructed as recommended (Quantum), with minor modifications.
  • cDNAs are expressed under the control of a cytomegalovirus promoter (CMV5) in each vector.
  • each adenoviral vector plasmid in which cDNA is ligated is transfected into human fetal kidney 293 cells (Microbix, Toronto, Canada); after 14- 21 days, homologous recombination occurs in the cells, leading to plaque formation. Plaques are isolated, and supernatants are eluted to infect 293 cells in 24-well culture plates. ⁇ eLa cells are infected to check transgene expression by immunoblot analysis and confocal microscopy for Gfp. After selection of viral clones, 293 cells are infected with individual clones for each vector to develop virus stocks. The viruses are purified by CsCl gradient centrifugation. Viral titers are determined by plaque assay, absorbance measurement, and serially diluted infection of GFP vector aliquots followed by confocal microscopic observation.
  • Viral supernatants from infected 293 cells are treated with proteinase K (10 ⁇ g/ml) and analyzed by PCR amplification of viral DNAs.
  • Cell pellets are treated with 1% SDS and proteinase K (10 ⁇ g/ml) prior to PCR amplification.
  • DNA sequencing reactions are performed by Applied Biosystems Prism BigDye terminator reaction chemistry on a Perkin- ⁇ lmer Gene Amp PCR system 9600 and the Applied Biosystems Prism 377 DNA sequencing systems.
  • SAB ⁇ cells are infected with viral supernatants and analyzed by flow cytometry to confirm that the vectors do not cause cytotoxicity.
  • a previous study showed that adenoviral-EH/P expression did not cause apoptosis nor alter cell growth in normal human bronchial epithelial cells. (25).
  • Adenoviral infection is performed with 3x10 ⁇ cells that had been cultured for 24 hours in 6-well culture plates. Cells are incubated with adenoviral aliquots at a desired MOI in a 37° C, CO2 incubator for 1 hour, followed by addition of culture medium (>25 x volume of viral sample).
  • 1x10 ⁇ cells are fixed with 70% ethanol for 10 minutes, incubated with RNase A, and stained with propidium iodide for flow cytometric analysis. (30). MTS assay is performed with a kit (Promega, Madison, WI), as recommended by the manufacturer. For cell growth kinetics, 1x10 ⁇ cells/well are cultured in 6-well culture plates. The number of cells per well is counted at indicated times in triplicate, excluding the dead cells by trypan blue staining.
  • Tumorigenicity Cells are inoculated subcutaneously into the left dorsal region of three 6 week- old male BALB/c nude mice in each experimental group. The tumor volume for each mouse is determined by measuring in two directions and calculated as: tumor volume
  • Immunoblot analysis is performed by standard protocols. (29). Briefly, cells are cultured in 6-well plates and lysed for 30 minutes on ice in 100 ⁇ l of lysis buffer. (29). Protein concentrations are determined by the BioRad microassay. Cell lysates are subjected to 4-12% linear gradient SDS-PAGE and are electroblotted to nitrocellulose membranes (BioRad, Hercules, CA).
  • the membranes are blocked with 5% skim milk and probed with rabbit polyclonal anti-Fhit (Zymed, South San Francisco, CA), rat monoclonal anti-caspase 8 (Zymed), monoclonal anti-PARP (Clontech), goat polyclonal anti-Bid (Santa Cruz, Santa Cruz, CA), rabbit polyclonal anti-Caspase 9 (Santa Cruz), monoclonal anti-actin (Santa Cruz), monoclonal anti- lacZ (Sigma, St. Louis, MO), and monoclonal anti-Gfp antibodies (Clontech) at recommended dilutions. After probing with an appropriate secondary antibody (Amersham, Piscataway, NJ), the signal is detected by the enhanced chemiluminescence system (Amersham). Immunohistochemical analysis with anti- human Fhit antibody is performed as described. (31).
  • Adenoviral Fhit expression in esophageal cancer cells in vitro Immunoblot analysis of protein from seven esophageal cancer cell lines, TE1, TE2, TE4, TE10, TE12, TE13, and TE14 shows that TE4 and TE10 cells express endogenous Fhit protein, while endogenous Fhit protein is undetectable in TE1, TE2, TE12, TE13, and TE14 cells.
  • Fig. 1A These seven esophageal cancer cell lines were infected with adenoviral-EH/P, adenoviral-EH/P-GEP, adenoviral-GEP, and adenoviral-LACZ vectors.
  • adenoviral-EH/P shows that adenoviral-EH/P, adenoviral-EH/P-GEP, adenoviral-GEP, and adenoviral-LACZ infections result in substantial expression of transgenes at 24 hours after infection, which persists for at least a week (transgene expression at 72 hours after infection is shown in Fig. IB).
  • Immunoblot analysis and Coomassie brilliant blue staining show that almost equal amounts of Fhit protein are expressed after infection with the same MOI of adenoviral-EH/P and adenoviral-EH/P-GEP vectors. (Fig. IB).
  • TE4 and TE14 cells show Fhit-induced apoptosis in a viral MOI-dependent manner.
  • the TE12 cell line shows cell population accumulation at the S to G2/ phase, accompanied by a small fraction of apoptotic cells, at 72 hours, which is much more evident at 5 days after infection with adenoviral-EH/P and adenoviral-EH/P-GEP vectors. There is little effect of infection with adenoviral-GEP and adenoviral-LACZ vectors.
  • T ⁇ 1, T ⁇ 2, TE10, and TE13 cells do not show obvious apoptosis or cell cycle arrest (Fig. 2A and B), although adenoviral Fhit protein is abundantly expressed (Fig. IB).
  • Fig. 5A These molecules are not, or are barely, activated in T ⁇ 1, T ⁇ 2, TE10, TE12, and TE13 cells after adenoviral-EH/P induction.
  • Fig. 5A PARP is cleaved in T ⁇ 4 and TE14 cells after adenoviral-EH/P transduction, but not after adenoviral-GEP infection, while uncleaved PARP is barely detected in T ⁇ 10 after adenoviral-GFP or adenoviral-EH/P infection.
  • PARP is not activated or is barely activated in T ⁇ 1, T ⁇ 2, TE12, and TE13 cells after adenoviral-EH/P induction.
  • Caspase 8 is cleaved in all seven esophageal cancer cells after adenoviral-EH/P transduction, but not adenoviral-GEP.
  • Fig. 5B adenoviral-EH/P-infected T ⁇ 4 and TE14 cells were cultured in medium with caspase inhibitors.
  • caspase inhibitors Z-NAD-fmk, Z-DEVD-fmk, or Z-IETD-fmk
  • flow cytometry analysis shows that apoptotic fractions are significantly inhibited.
  • FIG. 6A and B These data demonstrate that Fhit-induced apoptosis is controlled, at least in part, by caspase-dependent pathways.
  • MTS assay shows that in vitro cell growth of TE4 and TE14 cells treated with adenoviral-EH/P and adenoviral-EH/P-GEP is inhibited compared with control experiments using the adenoviral-GEP and the adenoviral-LACZ vectors.
  • Fig. 7A and B Cell counts reveal that in vitro growth of adenoviral-EH/P- or adenoviral- EH/P-GEP-infected T ⁇ 12 cells is inhibited compared with adenoviral-GEP- and adenoviral-LACZ-infected T ⁇ 12 cells.
  • Fig. 7C The flow cytometry (Fig.
  • T ⁇ 14 cells When inoculated in nude mice, T ⁇ 14 cells, but not TE4 and TE12 cells, are tumorigenic, as previously reported. (35).
  • 1 10 ⁇ TE14 cells are infected in vitro at MOI 30 with adenoviral-EH/P, adenoviral-EH/P-GEP, adenoviral-GEP, and adenoviral-LACZ vectors, cultured for 24 hours, and inoculated subcutaneously into nude mice, tumorigenicity of adenoviral-EH/P- or adenoviral-EH/P-GEP-infected T ⁇ 14 cells is reduced compared with adenoviral-GEP- and adenoviral-LACZ-infected T ⁇ 14 cells.
  • FIG. 8A and B Lrmunohistochemical analysis shows that Fhit protein is abundantly expressed in TE14 cells after adenoviral-EH/P and adenoviral -EH/P- GFP infection.
  • FIG. 8C Lrmunohistochemical analysis shows that Fhit protein is abundantly expressed in TE14 cells after adenoviral-EH/P and adenoviral -EH/P- GFP infection.
  • the data of the present invention demonstrate tumor suppression by Fhit protein in 3 of 7, or about 40%, of esophageal cancer cell lines.
  • adenoviral transduction of the FHIT gene product causes suppression of cell growth in vitro.
  • two esophageal cancer cell lines exhibit caspase-dependent apoptosis while another shows accumulation of cells at G2 M, with inhibition of cell growth accompanied by a small fraction of apoptotic cells.
  • Treatment with adenoviral-EH/P vectors also reduces the tumorigenicity of T ⁇ 14 cells in vivo.
  • adenoviral-GEP and adenoviral-LACZ do not cause alterations in cell cycle or cell growth, and adenoviral-EH/P expression minimally affects cell cycle and cell growth in T ⁇ 1, T ⁇ 2, TE10, and TE13 cells, in which the transgenes are abundantly expressed.
  • adenoviral-EH/P overexpression (MOI 10) does not effect cell growth in normal human bronchial epithelial cells. (25).
  • flow cytometry analysis does not show significant alteration of the cell cycle in normal SAB ⁇ cells after adenoviral- FHIT overexpression at MOI 30.
  • Adenoviral-EH/P expression causes significant reduction of cell growth in 3 of 7 cell lines, i.e., endogenous Fhit(-) T ⁇ 14, Fhit(-) TE12, and Fhit(+) TE4 cells.
  • Pro-apoptotic molecules such as caspase 9 and Bid (34) are cleaved in both T ⁇ 4 and TE14 cells, but are not, or are barely, cleaved in the other five cell lines after adenoviral-EH/P transduction.
  • Caspase 8 is cleaved in all seven esophageal cancer cells, specifically after adenoviral-EH/P transduction, implying that caspase 8 is downstream of Fhit in a signaling pathway in all the esophageal cancer cells.
  • adenoviral-EH/P transduction results in activation, not only of the mitochondrial pathway, but also of the caspase 8 pathway, possibly amplified through Bid cleavage (37); because caspase 8 is activated by FHIT overexpression in all seven cell lines, capase 8 activation may be downstream of Fhit, but upstream of caspase 9 activation and Bid and PARP cleavage.
  • the present invention relates to the treatment of cancer by inducing apoptosis in cancer cells.
  • the delivery of the FHIT gene will be effective in treating human cancers in which the loss of Fhit is implicated.
  • endogenous Fhit expression is altered not only in advanced esophageal carcinomas but even in precarcinomatous lesions. (20, 21).
  • in vivo experiments have demonstrated that inactivation of one Fhit allele in recombinant mice resulted in a much higher susceptibility to carcinogen-induced esophageal/forestomach cancer. (38).

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Abstract

Inactivation of FHIT, a tumor suppressor gene, and loss of Fhit protein expression is observed in many human cancers. The present invention provides a method for the treatment of cancers. Specifically, the invention provides a method for inducing apoptosis in cancer cells via introduction of a tumor suppressor gene into the cells. The present invention relates to the delivery of the FHIT gene by way of an adenoviral vector, or other DNA transport system, into the cancer cells of an individual. This invention has particular application to the treatment of esophageal cancer and other tumors that carry alterations of the FHIT gene.

Description

ADENO VIRAL TRANSDUCTION OF FRAGILE HISTIDINE TRIAD (FHIT) INTO CANCER CELLS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 60/268,097 filed February 12, 2001.
GOVERNMENT RIGHTS TO THE INVENTION
The invention was made in part with government support under grants CA77738, CA51083, CA21124, and CA56336 awarded by the National Cancer Institute. The government has certain rights to the invention.
FIELD OF THE INVENTION
This invention relates to the fields of molecular biology and-gene therapy and is directed to a method of treating cancer, and, more particularly, to inducing apoptosis in esophageal cancer cells by gene delivery using viral vectors expressing the Fragile Histidine Triad ("FHIT') gene.
BACKGROUND OF THE INVENTION
Human chromosome 3p is one of the chromosomal regions most frequently deleted in human tumors, including those of lung, breast, esophagus, and bladder. (1). Positional cloning of this region has led to identification of the FHIT gene, which encodes a member of the histidine triad protein superfamily. (2-5). The FHIT gene encompasses the FRA3B fragile site (2, 6), and a genomic locus which is frequently involved in allelic loss, genomic rearrangement, and cytogenetic abnormalities in tumors (2-4). Numerous studies have shown genomic alterations at the FHIT locus, such as biallelic deletions, translocations, and the loss of Fhit protein expression in many human cancers (2-4), including those of the esophagus, lung, breast, cervix, and bladder. While point mutations within the FHIT gene are rare (7-9), deletions are extremely common (2-4), and, less frequently, methylation is involved in Fhit inactivation (10). Nucleotide sequence analysis of the FHIT locus in tumor cell lines indicates that long interspersed nuclear element ("LINE") and long terminal repeal ("LTR") sequences are involved in homologous recombinations at the deletion endpoints in most cancers. (11, 12). Presumably because FHIT encompasses the fragile region, which is carcinogen sensitive, the FHIT gene is susceptible to damage caused by environmental carcinogens, leading to clonal expansion of Fhit negative cells.
Esophageal cancer, one of the most deadly human tumors, occurs worldwide, and its incidence is increasing in the Western world. (13, 14). Therapeutic approaches for esophageal cancer include not only conventional therapies, such as surgical removal and radiation treatment, but gene therapy strategies, such as introduction of the tumor suppressor pl6/LNK4 (15); the expression of LL2, IL6 and GM-CSF gene products (16, 17); and the transduction of the herpes simplex virus- thymidine kinase gene (18, 19). Previous studies have shown that Fhit expression is lost even in an early stage of esophageal carcinogenesis and have indicated a significant correlation with heavy smoking and alcohol habits (20, 21), providing the rationale for assessment of the biological effects of FHIT gene transduction in esophageal cancer cells.
Previous FHIT gene replacement experiments mainly involved stable transfectants of endogenous Fhit-negative tumor cells to assess the biological function of Fhit protein. Stable exogenous Fhit expression in Fhit-negative lung, gastric, and renal cancer cells resulted in inhibition of tumor cell growth (22-24), at least in part due to induction of apoptosis (23). Similarly, Ji et al. demonstrated that reintroduction of Fhit protein by adenoviral-EfflT gene transduction in lung and head- ' and-neck cancer cell lines caused apoptosis and inhibition of tumorigenicity. (25). Other studies have questioned the status of FHIT as a tumor suppressor, based on observations of tumorigenicity of stable FHIT transfectants. (26, 27). The present invention provides evidence that transient Fhit expression after adenoviral-EH/r transduction of esophageal cancer cells suppresses cancer cell growth in vitro. Additional evidence demonstrates that tumorigenicity is abrogated by adenoviral FHIT infection in esophageal cancer cell lines. The present invention is directed to a method of inhibiting the growth of cancer cells and tumor development through adenoviral transduction of FHIT.
ABBREVIATIONS
"FHIT" means "Fragile Histidine Triad"
"SABE" means "small air way bronchial epithelial"
"Z-NAD-fmk" means "benzyloxycarbonyl-valinyl-alaninyl-aspartyl(O- methyl)-fluoromethylketone "GFP" means "green fluorescent protein"
"MOI" means "multiplicity of infection
"MTS" means "[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium inner salt]"
"Νit" means "nitrilases"
DEFINITIONS
"cancer" is any disorder of the class of diseases of animals characterized by uncontrolled cellular growth, which disesase can be either premalignant or malignant.
SUMMARY OF THE INVENTION
It is an object of the present invention to induce apoptosis in mammalian cancer cells by transfenϊng a FHIT gene, or derivative thereof, to the cells. A nucleic acid encoding a FHIT gene, or derivative thereof, is delivered to the cancer cells. A Fhit protein, or derivative thereof, is expressed by the FHIT gene, or derivative thereof, in the mammalian cells, thereby inducing apoptosis. In one embodiment, the nucleic acid encoding the EHH gene, or derivative thereof, is in a vector. In another embodiment, the vector is a viral vector. In another embodiment, the viral vector is a recombinant adenovirus. In one embodiment of the present invention the mammalian cancer cells are esophageal cancer cells.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Adenoviral-EHZT expression in esophageal cancer cell lines.
(A) Immunoblot analysis of endogenous Fhit expression. Cell lysates (60 μg protein) are subjected to SDS-PAGΕ. Lane 1, esophageal cancer TΕ1 cell line; lane 2, TΕ2; lane 3, TE4; lane 4, TE10; lane 5, TE12; lane 6, TE13; and lane 7, TE14.
(B) Immunoblot analysis of adenoviral-EHH expression. Cell lysates (30 μg protein) are extracted at 72 hrs after infections at MOI 20 and subjected to SDS-PAGΕ.
Immunoblot analysis perforrned with anti-Fhit antibody (LB, top) and with anti- actin antibody (L3, bottom) is shown. Coomassie brilliant blue staining (CB) is shown in the middle. Adenoviral-EHiT (lanes 1-7) and adenoviral-EHZT-GEP (lanes 8-14) vectors are used for infection of esophageal cancer TΕ1 (lanes 1 and 8), TΕ2 (lanes 2 and 9), TE4 (lanes 3 and 10), TE10 (lanes 4 and 11), TE12 (lanes
5 and 12), TE13 (lanes 6 and 13), and TE14 (lanes 7 and 14) cells.
Figure 2. Flow cytometry of infected esophageal cancer cell lines.
Flow cytometry at 72 hours (A) and at 5 days (B) after infection with adenoviral-EH/E (the top row in each figure), adenoviral-EHH-GEP (the second row from the top), adenoviral-GEP (the third row), and adenoviral-LA CZ (the bottom row) at MOI 20.
Figure 3. Increased apoptosis of adenoviral-EHiT infected esophageal cancer cell lines.
Flow cytometry analysis of infected esophageal cancer cells. Apoptotic fractions are shown for TΕ4, TE10 and TE14 cells infected by adenoviral-EH/E- GFP (closed characters) and adenoviral-GEP (open characters) vectors over 48 hrs. The y- axis shows apoptotic fraction (%) determined by flow cytometry, whereas the x-axis indicates MOI.
Figure 4. Increased apoptosis of adeno viral -FHIT infected HeLa cells. To assess the effect of adenoviral Fhit expression in another cell type, the cervical cancer cell line, HeLa, is infected with viral vectors.
(A) Flow cytometry analysis of HeLa cells infected by adenoviral FHIT-GFP and adenoviral-GEP at MOI 50.
(B) Diagram depicting the infection of HeLa cells by adenoviral-EHiT- GFP (A) and adenoviral-GEP (Δ) vectors over 48 hrs.
(C) Immunoblot analysis of Fhit protein expression in adenoviral FHIT-GFP infected ΗeLa cells at MOI 50 (lane 1) and in control ΗeLa cells (lane 2). Analysis is performed with Fhit antibody (upper lanes) and with anti-actin antibody (lower lanes)
Figure 5. Activation of proapoptotic proteins in adenoviral-EHH infected esophageal cancer cell lines.
(A) Immunoblot analysis of PARP, Bid, and caspase 9 in TΕ1 (lane 1), TΕ2 (lane 2), TE4 (lanes 3, 8 and 9), TE10 (lane 4), TE12 (lanes 5, 10 and 11), TE13 (lane 6), and TE14 (lanes 7, 12 and 13) cells infected with adenoviral-EH/T
(lanes 1-8, 10 and 12) or adenoviral-GEP (lanes 9, 11 and 13) at MOI 20 for 72 hours. Cell lysates (50 μg protein) are subjected to SDS-PAGΕ. Immunoblot analysis is performed with PARP, Bid, caspase 9, and β-actin antibodies as indicated. Precursor forms of PARP, Bid, and procaspase 9 are cleaved in TΕ4 and TE14 cells.
(B) Immunoblot analysis of caspase 8 in TE4 (lanes 1 and 5), TE10 (lanes 2 and 6), TE12 (lanes 3 and 7), and TE14 (lanes 4 and 8) cells infected with adenoviral-EH/P (lanes 1-4) or adenoviral-GEP (lanes 5-8) at MOI 20 for 72 hours. Activation of procaspase 8 results in a cleaved form of caspase 8. Analysis is performed with caspase 8 and β-actin antibodies as indicated. Figure 6. Culture with caspase inhibitors in adenoviral-EH/P infected esophageal cancer cell lines.
Flow cytometry analysis of TΕ4 (A) and TE14 (B) cells infected with adenoviral-EH/r at MOI 30 for 72 hours in medium with caspase inhibitors, Z-VAD- fmk, Z-DΕVD-fmk, and Z-IΕTD-fmk as indicated
Figure 7. In vitro cell growth of esophageal cancer cell lines after adenoviral-EHH expression.
(A, B) MTS assay of TΕ14 (A) and TE4 (B) cells infected with adenoviral-EH/P (•), adenoviral-EH/E-GEP (■), adenoviral-GEP (O) and adenoviral- A CZ (D) vectors at MOIs 30, 10, 5, and 2 for the indicated periods. Culture medium is renewed daily. Data on infection of TΕ4 cells at MOI 30 are shown. Results are shown as an inhibition (%) by comparison with non-infected control experiments. Each data point shown is an average of four independent assays. (C) Growth curve for TE12 cells. The cell number is counted by trypan blue exclusion at the indicated times after infection with adenoviral-EH/P (•), adenoviral-EH/P-GEP (■), adenoviral-GEP (O), and adenoviral- ACZ (D) vectors.
Figure 8. Tumorigenicity of adenoviral EH/T-transduced TΕ14 cells^
(A) Tumor sizes in nude mice. TE14 cells are infected by adenoviral-EH/P (•), adenoviral-EH/P-GEP (■), adenoviral-GEP (O), and adenoviral-LACZ vector
(D) as described in the text. Cells are injected subcutaneously into nude mice and tumor sizes measured. (B) Tumors formed in nude mice. Tumors were excised on day 18 after injection. The three tumors at the top are from TΕ14 cells infected with adenoviral- FHIT, the three in the second row are from TE14 cells infected with adenoviral-EHH-GEP, the three in the third row are from TΕ14 cells infected with adenoviral-GEP, and the three in the bottom row are from TΕ14 cells infected with adenoviral-LACZ. A scale bar, 10 mm. (C) Immunohistochemical analysis of excised tumor tissues. The tissues are stained with anti-human Fhit antibody. I, tumor infected with adenoviral- FHIT (upper left; magnification, x400); II, tumor infected with adenoviral- LACZ (upper right; magnification, x250); III, tumor infected with adenoviral- GFP (lower left; magnification, x250); and IV, tumor infected with adenoviral-EHTP-GEP (lower right; magnification, x400).
DETAILED DESCRIPTION OF THE INVENTION
The FHIT gene has been identified in a region at chromosome 3pl4.2 that is deleted in many tumors, including esophageal cancer. Genetic alterations of the FHIT gene are frequently observed in many human cancers, and a tumor suppressor function of Fhit has been demonstrated. In the present invention, the biological effects of adenoviral-EH/P transduction in esophageal cancer cell lines is examined.
Materials and Methods Cell Culture The cervical cancer cell line, ΗeLa, and esophageal cancer cells, TΕ1, TΕ2, TE4, TE10, TE12, TE13, and TE14 are maintained in RPMI 1640 medium with 10% fetal bovine serum. Cells can be those of any animal, including, but not limited to, animals such as cows, pigs, horses, etc., and are preferably those of a mammal, and most preferably human. SABE cells are obtained from Clonetics, Walkersville, MD, and cultured as recommended. The following caspase inhibitors are purchased from Calbiochem (San Diego, CA): Z-VAD-fmk, an inhibitor for caspases 1, 3, 4 and 7; Z-DEND-fmk, an inhibitor for caspases 3, 6, 7, 8 and 10; and Z-IETD-fmk, an inhibitor for caspase 8.
Recombinant adenoviral vector construction and gene transduction Full-length FHIT cDΝA is isolated from normal human placenta cDΝA
(Clontech) by RT-PCR strategy and confirmed by DΝA sequencing. (2). cDΝAs for Gfp and lacZ are obtained from expression vectors (Clontech, Palo Alto, CA). Each cDΝA is ligated into an adenoviral backbone DΝA (Quantum, Montreal, Canada). Four adenoviral vectors, an adenoviral-EH/P-GEP vector that encodes two separate proteins through the internal ribosome entry site, an adenoviral-EH/P vector, an adenoviral-GEP vector, and an adenoviral-LACZ vector are constructed as recommended (Quantum), with minor modifications. (25, 28). cDNAs are expressed under the control of a cytomegalovirus promoter (CMV5) in each vector.
Briefly, each adenoviral vector plasmid in which cDNA is ligated is transfected into human fetal kidney 293 cells (Microbix, Toronto, Canada); after 14- 21 days, homologous recombination occurs in the cells, leading to plaque formation. Plaques are isolated, and supernatants are eluted to infect 293 cells in 24-well culture plates. ΗeLa cells are infected to check transgene expression by immunoblot analysis and confocal microscopy for Gfp. After selection of viral clones, 293 cells are infected with individual clones for each vector to develop virus stocks. The viruses are purified by CsCl gradient centrifugation. Viral titers are determined by plaque assay, absorbance measurement, and serially diluted infection of GFP vector aliquots followed by confocal microscopic observation.
Potential contamination with wild-type virus is monitored by PCR analysis (Quantum). Viral supernatants from infected 293 cells are treated with proteinase K (10 μg/ml) and analyzed by PCR amplification of viral DNAs. Cell pellets are treated with 1% SDS and proteinase K (10 μg/ml) prior to PCR amplification. DNA sequencing reactions are performed by Applied Biosystems Prism BigDye terminator reaction chemistry on a Perkin-Εlmer Gene Amp PCR system 9600 and the Applied Biosystems Prism 377 DNA sequencing systems. SABΕ cells are infected with viral supernatants and analyzed by flow cytometry to confirm that the vectors do not cause cytotoxicity. A previous study showed that adenoviral-EH/P expression did not cause apoptosis nor alter cell growth in normal human bronchial epithelial cells. (25).
Adenoviral infection is performed with 3x10^ cells that had been cultured for 24 hours in 6-well culture plates. Cells are incubated with adenoviral aliquots at a desired MOI in a 37° C, CO2 incubator for 1 hour, followed by addition of culture medium (>25 x volume of viral sample).
Flow cytometry, MTS assay and cell counting Flow cytometry analysis is performed by standard protocols. (29, 30).
Briefly, 1x10^ cells are fixed with 70% ethanol for 10 minutes, incubated with RNase A, and stained with propidium iodide for flow cytometric analysis. (30). MTS assay is performed with a kit (Promega, Madison, WI), as recommended by the manufacturer. For cell growth kinetics, 1x10^ cells/well are cultured in 6-well culture plates. The number of cells per well is counted at indicated times in triplicate, excluding the dead cells by trypan blue staining.
Tumorigenicity Cells are inoculated subcutaneously into the left dorsal region of three 6 week- old male BALB/c nude mice in each experimental group. The tumor volume for each mouse is determined by measuring in two directions and calculated as: tumor volume
= length x (width)2/2. (25).
Immunoblot analysis and immunohisto chemistry
Immunoblot analysis is performed by standard protocols. (29). Briefly, cells are cultured in 6-well plates and lysed for 30 minutes on ice in 100 μl of lysis buffer. (29). Protein concentrations are determined by the BioRad microassay. Cell lysates are subjected to 4-12% linear gradient SDS-PAGE and are electroblotted to nitrocellulose membranes (BioRad, Hercules, CA). The membranes are blocked with 5% skim milk and probed with rabbit polyclonal anti-Fhit (Zymed, South San Francisco, CA), rat monoclonal anti-caspase 8 (Zymed), monoclonal anti-PARP (Clontech), goat polyclonal anti-Bid (Santa Cruz, Santa Cruz, CA), rabbit polyclonal anti-Caspase 9 (Santa Cruz), monoclonal anti-actin (Santa Cruz), monoclonal anti- lacZ (Sigma, St. Louis, MO), and monoclonal anti-Gfp antibodies (Clontech) at recommended dilutions. After probing with an appropriate secondary antibody (Amersham, Piscataway, NJ), the signal is detected by the enhanced chemiluminescence system (Amersham). Immunohistochemical analysis with anti- human Fhit antibody is performed as described. (31).
Results
Adenoviral Fhit expression in esophageal cancer cells in vitro Immunoblot analysis of protein from seven esophageal cancer cell lines, TE1, TE2, TE4, TE10, TE12, TE13, and TE14 shows that TE4 and TE10 cells express endogenous Fhit protein, while endogenous Fhit protein is undetectable in TE1, TE2, TE12, TE13, and TE14 cells. (Fig. 1A). These seven esophageal cancer cell lines were infected with adenoviral-EH/P, adenoviral-EH/P-GEP, adenoviral-GEP, and adenoviral-LACZ vectors. Immunoblot analysis shows that adenoviral-EH/P, adenoviral-EH/P-GEP, adenoviral-GEP, and adenoviral-LACZ infections result in substantial expression of transgenes at 24 hours after infection, which persists for at least a week (transgene expression at 72 hours after infection is shown in Fig. IB). Immunoblot analysis and Coomassie brilliant blue staining show that almost equal amounts of Fhit protein are expressed after infection with the same MOI of adenoviral-EH/P and adenoviral-EH/P-GEP vectors. (Fig. IB).
Cell cycle analysis of FHIT -transduced esophageal cancer cell lines Flow cytometry analysis of the seven esophageal cancer cell lines infected with adenoviral-EH/P, adenoviral-EH/P-GEP, and control vectors shows that FHIT transduction induces increased apoptotic cell populations in two cell lines, TΕ4 and TE14, while control vectors induce little or no apoptosis, at 72 hours and 5 days after infection. (Fig. 2A and B). At 72 hours after infection (Fig. 2A) with adenoviral- FHIT and adenoviral-EH/P-GEP vectors, 23% (adenoviral-EH/P) and 25% (adenoviral-EH/P-GEP) of TΕ4, and 39% (FHIT) and 25% (FHIT-GFP) of TE14 cells have undergone apoptosis, whereas the fraction of apoptotic cells increases to 53% (FHIT) and 42% (FHIT-GFP) of TE4, and 46% (FHIT) and 45% (FHIT-GFP) of TE14 cells at 5 days. (Fig. 2B). TE4 and TE14 cells show Fhit-induced apoptosis in a viral MOI-dependent manner. (Fig. 3). The TE12 cell line shows cell population accumulation at the S to G2/ phase, accompanied by a small fraction of apoptotic cells, at 72 hours, which is much more evident at 5 days after infection with adenoviral-EH/P and adenoviral-EH/P-GEP vectors. There is little effect of infection with adenoviral-GEP and adenoviral-LACZ vectors. (Fig. 2). TΕ1, TΕ2, TE10, and TE13 cells do not show obvious apoptosis or cell cycle arrest (Fig. 2A and B), although adenoviral Fhit protein is abundantly expressed (Fig. IB).
To assess another tumorigenic cell type that expresses a low level of endogenous Fhit, a cervical cancer cell, ΗeLa, was infected with the adenoviral FHIT vector to determine the effect of Fhit overexpression. The results show Fhit-induced apoptosis of HeLa cells in a viral MOI-dependent manner. (Fig. 4).
Analysis of adenoviral FHIT-induced apoptosis Immunoblot analyses with antibodies against caspases 8 and 9, Bid, and PARP on lysates from esophageal cancer cell lines before and after infections are compared to determine if major mediators of apoptosis (32-34) are involved in adenoviral FHIT- induced cell death. Cleavage of Bid and caspase 9 in TE4 and TE14 cells after adenoviral-EH/P transduction, but not after adenoviral-GEP infection is observed. (Fig. 5) These molecules are not, or are barely, activated in TΕ1, TΕ2, TE10, TE12, and TE13 cells after adenoviral-EH/P induction. (Fig. 5A). PARP is cleaved in TΕ4 and TE14 cells after adenoviral-EH/P transduction, but not after adenoviral-GEP infection, while uncleaved PARP is barely detected in TΕ10 after adenoviral-GFP or adenoviral-EH/P infection. PARP is not activated or is barely activated in TΕ1, TΕ2, TE12, and TE13 cells after adenoviral-EH/P induction. (Fig. 5A). Caspase 8 is cleaved in all seven esophageal cancer cells after adenoviral-EH/P transduction, but not adenoviral-GEP. (Fig. 5B). These data show involvement of caspase pathways in Fhit-induced apoptosis. To confirm this, adenoviral-EH/P-infected TΕ4 and TE14 cells were cultured in medium with caspase inhibitors. When TE4 and TE14 cells are cultured with caspase inhibitors, Z-NAD-fmk, Z-DEVD-fmk, or Z-IETD-fmk, flow cytometry analysis shows that apoptotic fractions are significantly inhibited. (Fig. 6A and B). These data demonstrate that Fhit-induced apoptosis is controlled, at least in part, by caspase-dependent pathways. (34).
Cell growth analysis of adenoviral FHIT-infected esophageal cancer cell lines in vitro
MTS assay shows that in vitro cell growth of TE4 and TE14 cells treated with adenoviral-EH/P and adenoviral-EH/P-GEP is inhibited compared with control experiments using the adenoviral-GEP and the adenoviral-LACZ vectors. (Fig. 7A and B). Cell counts reveal that in vitro growth of adenoviral-EH/P- or adenoviral- EH/P-GEP-infected TΕ12 cells is inhibited compared with adenoviral-GEP- and adenoviral-LACZ-infected TΕ12 cells. (Fig. 7C). The flow cytometry (Fig. 2) and cell growth data imply that adenoviral-EH/P expression results in cell cycle arrest in TE12 cells, a response reminiscent of lung cancer cell cycle arrest and accumulation in S phase after adenoviral FHIT infection. (25). Growth of TE1, TE2, TE10, and TE13 cells shows no significant alteration after adenoviral-EH/P and control vector infection.
Tumorigenicity of adenoviral infected esophageal cancer cells
When inoculated in nude mice, TΕ14 cells, but not TE4 and TE12 cells, are tumorigenic, as previously reported. (35). When 1 10^ TE14 cells are infected in vitro at MOI 30 with adenoviral-EH/P, adenoviral-EH/P-GEP, adenoviral-GEP, and adenoviral-LACZ vectors, cultured for 24 hours, and inoculated subcutaneously into nude mice, tumorigenicity of adenoviral-EH/P- or adenoviral-EH/P-GEP-infected TΕ14 cells is reduced compared with adenoviral-GEP- and adenoviral-LACZ-infected TΕ14 cells. (Fig. 8A and B). Lrmunohistochemical analysis shows that Fhit protein is abundantly expressed in TE14 cells after adenoviral-EH/P and adenoviral -EH/P- GFP infection. (Fig. 8C).
Discussion
The data of the present invention demonstrate tumor suppression by Fhit protein in 3 of 7, or about 40%, of esophageal cancer cell lines. In 3 of 7 esophageal cell lines, adenoviral transduction of the FHIT gene product causes suppression of cell growth in vitro. And, after adenoviral-EH/P transduction, two esophageal cancer cell lines exhibit caspase-dependent apoptosis while another shows accumulation of cells at G2 M, with inhibition of cell growth accompanied by a small fraction of apoptotic cells. Treatment with adenoviral-EH/P vectors also reduces the tumorigenicity of TΕ14 cells in vivo. These data demonstrate tumor suppression by Fhit protein in esophageal cancer cell lines. Generalized toxicity of the viral vectors is ruled out because the control viruses, adenoviral-GEP and adenoviral-LACZ, do not cause alterations in cell cycle or cell growth, and adenoviral-EH/P expression minimally affects cell cycle and cell growth in TΕ1, TΕ2, TE10, and TE13 cells, in which the transgenes are abundantly expressed. In addition, a previous study showed that adenoviral-EH/P overexpression (MOI 10) does not effect cell growth in normal human bronchial epithelial cells. (25). Similarly, flow cytometry analysis does not show significant alteration of the cell cycle in normal SABΕ cells after adenoviral- FHIT overexpression at MOI 30. These findings imply that FHIT transduction will serve as a novel cancer therapeutic strategy.
Adenoviral-EH/P expression causes significant reduction of cell growth in 3 of 7 cell lines, i.e., endogenous Fhit(-) TΕ14, Fhit(-) TE12, and Fhit(+) TE4 cells. These findings indicate that susceptibility to apoptosis or cell growth inhibition is not restricted to cancer cells with a complete loss of Fhit expression. This is consistent with a recent analysis involving stable transfectants of renal carcinoma cells, which showed that susceptibility to suppression by exogenous Fhit expression is dependent on cell type and is not restricted to cancer cells without endogenous Fhit. (24). Two other studies reported that ΗeLa cells stably expressing exogenous Fhit exhibit no significant alteration in cell growth. (26, 27). In the present invention, when Fhit protein is expressed in ΗeLa cells by adenoviral-EH/P or adenoviral-EH/P-GEP infection, ΗeLa cells show marked apoptosis in each experiment, but not in a control experiment with GFP vectors. This observation implies that the threshold of Fhit expression necessary for biological effect may differ in individual cell types.
Although all the esophageal cancer cell lines in this study express very high levels of exogenous Fhit protein after infection, four of these cancer cell lines are insensitive to FHIT overexpression. One explanation might be that another gene(s) or protein(s) in the Fhit pathway is lost or inactivated in these cancer cells. Previous studies have shown that EH/Phomologs are encoded as fusion proteins with nitrilases (Nit) in Drosophila melanogaster and Caenorhabditis elegans (5, 36), suggesting that the human Nit might act in the Fhit pathway. Immunoblot analysis with anti-Nit antiserum reveals that all seven esophageal cancer cell lines express the Nit protein.
Several observations implicate Fhit in pro-apoptotic pathways. Pro-apoptotic molecules, such as caspase 9 and Bid (34), are cleaved in both TΕ4 and TE14 cells, but are not, or are barely, cleaved in the other five cell lines after adenoviral-EH/P transduction. Caspase 8 is cleaved in all seven esophageal cancer cells, specifically after adenoviral-EH/P transduction, implying that caspase 8 is downstream of Fhit in a signaling pathway in all the esophageal cancer cells. When adenoviral-EH/P infected TΕ4 and TE14 cells are cultured in medium with individual caspase inhibitors, protection from apoptosis is observed in each, implying that full execution of the Fhit-induced apoptosis requires both initiators, caspase 8 and 9. (34). Although no significant changes are observed in Bcl-2 and Bcl-XL expression in Fhit- associated apoptosis, Bid is cleaved in both TE4 and TE14 cells after adenoviral- EH P transduction, implying that Bid activation also is required for the onset of apoptosis. These data imply that adenoviral-EH/P transduction results in activation, not only of the mitochondrial pathway, but also of the caspase 8 pathway, possibly amplified through Bid cleavage (37); because caspase 8 is activated by FHIT overexpression in all seven cell lines, capase 8 activation may be downstream of Fhit, but upstream of caspase 9 activation and Bid and PARP cleavage.
The present invention relates to the treatment of cancer by inducing apoptosis in cancer cells. Specifically, the delivery of the FHIT gene will be effective in treating human cancers in which the loss of Fhit is implicated. Several recent studies have shown that endogenous Fhit expression is altered not only in advanced esophageal carcinomas but even in precarcinomatous lesions. (20, 21). Moreover, in vivo experiments have demonstrated that inactivation of one Fhit allele in recombinant mice resulted in a much higher susceptibility to carcinogen-induced esophageal/forestomach cancer. (38).

Claims

What is claimed is:
1. A method of inducing apoptosis in mammalian cancer cells, the method comprising a) delivering to said cells a nucleic acid encoding a FHIT gene, or derivative thereof; b) expressing a Fhit protein, or derivative thereof, encoded by said FHIT gene, or derivative thereof, in said cells; and c) inducing apoptosis.
2. The method of Claim 1 wherein said nucleic acid encoding a FHIT gene, or derivative thereof, is in a vector.
3. The method of Claim 2 wherein said vector comprises a viral vector.
4. The method of Claim 3 wherein said vector is a recombinant adenovirus.
5. The method of Claim 1 wherein said mammalian cancer cells are esophageal cancer cells.
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EP1272224A1 (en) * 2000-03-31 2003-01-08 Gene Logic, Inc. Gene expression profiles in esophageal tissue
EP1272224A4 (en) * 2000-03-31 2004-09-29 Gene Logic Inc Gene expression profiles in esophageal tissue
US8349560B2 (en) 2007-06-15 2013-01-08 The Ohio State University Research Method for diagnosing acute lymphomic leukemia (ALL) using miR-222
US9085804B2 (en) 2007-08-03 2015-07-21 The Ohio State University Research Foundation Ultraconserved regions encoding ncRNAs
EP2190979A1 (en) * 2007-08-31 2010-06-02 Aeras Global TB Vaccine Foundation Enhancement of transgene expression from viral-based vaccine vecors by expression of suppressors of the type i interferon response
EP2190979A4 (en) * 2007-08-31 2011-08-24 Aeras Global Tb Vaccine Foundation Enhancement of transgene expression from viral-based vaccine vecors by expression of suppressors of the type i interferon response
US8911998B2 (en) 2007-10-26 2014-12-16 The Ohio State University Methods for identifying fragile histidine triad (FHIT) interaction and uses thereof
US9125923B2 (en) 2008-06-11 2015-09-08 The Ohio State University Use of MiR-26 family as a predictive marker for hepatocellular carcinoma and responsiveness to therapy
US8916533B2 (en) 2009-11-23 2014-12-23 The Ohio State University Materials and methods useful for affecting tumor cell growth, migration and invasion
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US10758619B2 (en) 2010-11-15 2020-09-01 The Ohio State University Controlled release mucoadhesive systems
US11679157B2 (en) 2010-11-15 2023-06-20 The Ohio State University Controlled release mucoadhesive systems
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US9249468B2 (en) 2011-10-14 2016-02-02 The Ohio State University Methods and materials related to ovarian cancer
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US9434995B2 (en) 2012-01-20 2016-09-06 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis

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