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[Cancer Research 59, 4247-4251, September 1, 1999]
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[Cancer Research 59, 4247-4251, September 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Wild-Type p53 Induction Mediated by Replication-deficient Adenoviral Vectors1

Christina R. McPake2, Sheetal Shetty3, Geoffrey R. Kitchingman and Linda C. Harris4

Departments of Molecular Pharmacology [C. R. M., S. S., L. C. H.] and Cell and Gene Therapy [G. R. K.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Replication-deficient E1-/E3-deleted adenoviral vectors are commonly used to introduce transgenes into cells in vitro and have been used for certain kinds of gene therapy protocols in vivo. We have demonstrated that transduction of cells using these vectors can induce p53 expression in cells containing a wild-type p53 gene. This response is different from p53 induction observed after DNA damage because the time course of induction is slower and because it occurs in cells with an attenuated DNA damage response. However, this vector-induced p53 is transcriptionally active and, therefore, p53 function is not inactivated by viral proteins. The mechanism of induction appears to be an increased rate of protein translation because immunoprecipitation analyses demonstrated increased levels of 35S-labeled p53 protein, even after a short 15-min labeling time. Induction of p53 by adenoviral vectors may have various effects on transduced cells, including apoptosis and altered chemotherapy chemosensitivity. Therefore, the influence of the vector might confound the impact of any particular gene used in a gene therapy application.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The p53 gene encodes a phosphoprotein that is involved in many cellular processes, including inhibition of cell growth, apoptosis, and inhibition of transformation and genetic instability (1) . DNA tumor viruses, such as adenovirus, have developed mechanisms to inhibit cellular p53 function (2 , 3) . Host-induced apoptosis of infected cells prior to virus replication effectively inhibits the spread of infection. Therefore, it is essential for the virus to have a mechanism by which p53 can be inactivated, such that apoptosis is not initiated prior to completion of the viral replication cycle (4) . The adenoviral proteins that have been shown to play a role in the inhibition of p53 function are the early genes E1A, E1B, and E4. The E1A gene is primarily responsible for the immortalizing properties of adenoviruses (5) . This is mediated in part by the ability of E1A to bind to the product of the retinoblastoma gene, Rb (6) and also by its role in the stabilization of p53 (7) . The E1B gene also has transforming properties. It encodes two proteins, E1B Mr 55,000, which can directly bind to and inactivate p53 (8) , and E1B Mr 19,000, which inhibits initiation of DNA degradation induced by E1A (9) . The adenovirus E4 region also encodes a protein, the Mr 34,000 product of open reading frame 6, which binds to and inhibits the activity of p53 (10 , 11) .

The adenoviral vector used in these studies is derived from adenovirus of serotype 5 (Ad 5) and has most of the E1 region and all of E3 deleted. This type of vector, known as Av1, is the most common of the adenoviral vectors used today in gene therapy clinical trials and can transduce but not replicate in a wide variety of human cell types (12 , 13) . Using such a vector containing no inserted transgene, we have made the observation that transcriptionally active wtp535 can be induced in cells after virus transduction.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines.
NB-1643 and NB-1691 neuroblastoma cell lines were obtained from the Pediatric Oncology Group (St. Jude Children’s Research Hospital), and Rh30 and Rh18 cell lines were derived by and obtained from Dr. Peter Houghton (St. Jude Children’s Research Hospital). These lines were all grown in RPMI 1640 cell culture medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (Hyclone, Logan, UT). IMR90 lung fibroblasts were obtained from American Type Culture Collection (Manassas, VA) and grown in MEM (BioWhittaker) supplemented with 10% FCS (Hyclone). All cell lines were grown at 37°C in a humidified atmosphere of 5% CO2/95% air.

Adenoviral Vector.
The adenoviral vector used in these studies is derived from adenovirus of serotype 5 (Ad 5) and has most of the E1 region and all of E3 deleted. For these vectors to replicate E1A and E1B, proteins have to be supplied exogenously, for example, by infecting a complementing cell line such as 293 (12) , which expresses these proteins. The E3 region encodes proteins responsible for evading the host immune system (14) . Therefore, deletion of this region is a safety feature, ensuring easy detection and elimination by the host. Av1 was developed by Trapnell (12) .

A plasmid (pAVS6.DNA, obtained from Genetic Therapy Inc., Gaithersburg, MD) containing an expression cassette consisting of the RSV promoter and SV40 polyadenylation signal but no inserted transgene was homologously recombined with Ad-dl327 adenoviral DNA after transfection into 293 cells by standard methodology (15) . Plaque purified recombinant virus (Ad-VC) was determined to be free from wild-type virus contamination by PCR analysis for the E1A gene (16) . This assay results in a sensitivity of at least 10-9 (16) . Ad-VC was originally developed to be used as a control in studies in which results would be comparable with those obtained from similar vectors containing transgenes expressed under the control of the RSV promoter (17) . Ad-VC was used at mois ranging from 1 to 50 plaque-forming units/cell. Initially transduction was for 2 h in small volume of 2% serum containing media to just cover the cells (equivalent to 2 ml in a T75 flask). Complete medium containing 10% serum was then added (equivalent to 12 ml in a T75 flask). The cells were exposed to the adenovirus for a total of 24 h, at which time the medium was replaced. Cells which had been treated in a similar manner without addition of the virus were harvested as "time 0" samples. Any heat inactivation of virus was carried out for 1 h at 56°C.

Ad-ß-gal (obtained from Genetic Therapy Inc.) was developed by the same methodology as Ad-VC, which allows expression of the ß-galactosidase under the control of the RSV promoter after cell transduction.

Antibodies.
The p53 monoclonal antibody DO-1, conjugated to HRP (Santa Cruz Biotechnology, Santa Cruz, CA), was used at a concentration of 1:200. DO-1 that was not conjugated to HRP was used for the immunoprecipitation reactions. A p21 polyclonal antibody was obtained from Santa Cruz Biotechnology and used at a dilution of 1:500. A tubulin monoclonal antibody was obtained from Sigma Chemical Co. (St. Louis, MO) and used at a dilution of 1:1000. HRP-conjugated secondary antibodies were used when appropriate, either donkey antimouse IgG or donkey antirabbit IgG (Amersham, Arlington Heights, IL) at a dilutions of 1:1000 and 1:2000, respectively.

Western Analysis.
Extracts of soluble cellular protein were prepared by resuspending cell pellets in extract buffer [50 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 1 mM EDTA, 0.5 mM DTT, and 0.1% NP40] containing freshly added protease inhibitors; 10 µg/ml each antipain, aprotinin, and leupeptin; 1 mM sodium orthovanadate and 2 mM phenylmethylsulfonyl fluoride. Cells were then frozen and thawed three times in dry ice for 5 min followed by 37°C for 1 min. After microcentrifugation at 14,000 rpm at 4°C for 10 min, the pellets were discarded. Protein concentrations of all extracts were determined using the Bio-Rad protein determination kit (Bio-Rad Laboratories, Richmond, CA). Cell extracts were electrophoresed in 12.5% SDS-poyacrylamide gels, and Western analysis was performed as described previously (18) . A tubulin antibody was used as a loading control to confirm that equal amounts of protein had been loaded on to the gel.

Immunoprecipitation.
Cells plated into T162 flasks were radiolabeled with 120 µCi of [35S]methionine (1000 Ci/mmol; Amersham) in methionine-free medium at a concentration of 12 µCi/ml for 1 h or for 15 min. Cells were harvested immediately after removal of the isotope or 2 h after incubation in methionine-containing medium without isotope. Extracts (500 µg of each) were immunoprecipitated using p53 monoclonal antibody DO-1 (Santa Cruz Biotechnology). After incubation overnight with protein G PLUS-agarose beads (Santa Cruz Biotechnology), the immunoprecipitated p53 was electrophoresed on a 12% SDS-polyacrylamide gel, electroblotted to Immobilon-P membrane (Millipore, Bedford, MA), and exposed to X-ray film (Eastman Kodak Co., Rochester, NY). After a 2-week exposure the film was developed, and the bands were quantitated using Microsoft ImageQuant software. The same membrane was then exposed to p53-DO-1 HRP-conjugated antibody (Santa Cruz Biotechnology) to detect total cellular p53, as described above.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Initial experiments used NB-1643, a neuroblastoma cell line that expresses wtp53, which can be induced after exposure to ionizing radiation (Fig. 1A)Citation . As expected, DNA damage-induced p53 led to induction of p21, demonstrating transcriptionally active p53. Ad-VC at a moi of 1 was used to transduce these cells for 24 h, after which time fresh medium was applied. Fig. 1ACitation shows Western analysis of a time course of p53 and p21 induction after Ad-VC transduction. Although both ionizing radiation and exposure to Ad-VC induced transcriptionally active p53, the time course of induction was different. Induced p53 could be detected within 4 h postradiation, whereas it took 72 h to detect maximal induction of p53 expression after viral transduction. This difference suggests that these two stimuli, radiation and adenoviral vector transduction, induce p53 by two separate mechanisms or pathways. Exposure to higher mois resulted in a dose-dependent increase in p53 expression (Fig. 2A)Citation . A 2–3-fold induction was observed at a moi of 1, which can be increased to a 6-fold induction at a moi of 10 and an 8-fold induction at a moi of 50 (Fig. 2A)Citation . A more detailed time course at a moi of 10 revealed that Ad-VC-mediated p53 induction could be observed between 8 and 16 h after exposure to the virus (Fig. 2B)Citation . Ad-VC-mediated p53 induction was not observed within 4 h, and therefore, results were still in contrast to p53 induction after radiation exposure, even when cells were exposed to a higher dose of virus (Fig. 1)Citation .



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  		Fig. 1. Western analysis of p53 and p21 expression in NB-1643 cells (wtp53 gene) after exposure to either Ad-VC or ionizing radiation. Ad-VC exposure was at a moi of 1 for 24 h. The dose of ionizing radiation used was an IC80 dose of 175 rad. B, Western analysis of p21 expression in IMR90 normal human fibroblasts (wtp53 gene) after exposure to either Ad-VC or an IC80 dose (300 rad) of ionizing radiation.

 


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  		Fig. 2. Western analysis for p53 and p21 expression in NB-1643 cells. A, after exposure to increasing mois of Ad-VC for 24 h. B, after exposure to Ad-VC at a moi of 10 for increasing time intervals.

 
Fig. 1BCitation contains data derived from extracts of the normal human fibroblast cell line, IMR90, after exposure to ionizing radiation and Ad-VC. As in the NB-1643 cells, Fig. 1BCitation demonstrates that radiation-mediated p21 induction is evident much sooner than that induced after viral transduction. In this line, Ad-VC induced maximal expression at 24 h after transduction, whereas radiation-induced p21 expression occurs within 4 h of exposure. These data demonstrate that p53 can be induced by adenoviral vectors in nontransformed cells in addition to tumor cell lines.

We also examined three cell lines with attenuated endogenous p53 function (either MDM2 amplification or mutant p53) to determine whether these results were dependent upon a wtp53 gene sequence. Because it is the induction of functional wtp53 protein that is of interest and not simply increased protein levels due to stabilization of nonfunctional protein, only the results of p21 expression levels are shown. Fig. 3ACitation shows p21 induction after Ad-VC transduction in two cell lines with not only a wtp53 gene but also an amplified MDM2 gene (a neuroblastoma line NB-1691 and a rhabdomyosarcoma line, Rh18; Ref. 18 ). Overexpression of MDM2 protein leads to inactivation of p53 function because of the binding of MDM2 to the transactivation domain of p53 (19) . p53 and p21 cannot be induced after exposure to an IC80 dose of ionizing radiation in NB-1691 cells, whereas Rh18 cells do still demonstrate a DNA-damage response (Fig. 3B)Citation . However, both of these lines can induce p53-dependent p21 expression after Ad-VC transduction in a similar manner as described for NB-1643 and IMR90 cells (Fig. 3A)Citation . We have confirmed that Ad-VC transduction does not affect MDM2 binding to p53 (data not shown).



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  		Fig. 3. Western analysis of p21 expression after Ad-VC transduction of NB-1691 and Rh18 cells, which both express a wtp53 gene together with overexpression of MDM2. Rh30 cells express mutant p53 protein (codon 273). Measurement of tubulin expression was used as a control for equal protein loading. B, Western analysis demonstrating p53 and p21 expression 4 h after exposure to an IC80 dose of ionizing radiation (NB-1691, 780 rad; Rh18, 625 rad; Rh30, 725 rad).

 
The third line analyzed was Rh30, a rhabdomyosarcoma cell line expressing a mutant p53 gene (18) . As expected, neither p53 nor p21 could be induced after exposure to either ionizing radiation (Fig. 3B)Citation or Ad-VC transduction (Fig. 3A)Citation , confirming that the induction observed in the other lines was dependent upon a wtp53 gene sequence. We confirmed that Rh30 cells could be transduced by this kind of adenoviral vector by the use of a similar vector containing the wtp53 gene (Ad-p53; Genetic Therapy Inc.). Expression of functional exogenous wtp53 was confirmed by analysis of p21 expression in these cells after Ad-p53 transduction (data not shown).

It has been demonstrated that PI3K is activated after adenovirus receptor binding and that PI3K-activated signal transduction pathways are required for adenovirus internalization (20) . We investigated the possibility that adenovirus induced p53 could be mediated by PI3K activation by using wortmannin, an inhibitor of this kinase. Fig. 4Citation shows Western analysis of p53 and p21 24 h after the addition of Ad-VC (moi of 10) and wortmannin (300 nM), a dose previously shown to inhibit PI3K function (20) . Results demonstrated that addition of wortmannin had no effect on p53 or p21 expression after Ad-VC transduction (Fig. 4)Citation , and therefore, the PI3K pathway does not appear to be involved in the mechanism of induction. To confirm that wortmannin had no effect on adenovirus transduction, we used an Ad-ß-gal vector to transduce NB-1643 cells and confirmed that exogenous ß-galactosidase expression was unaffected by addition of wortmannin (data not shown).



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  		Fig. 4. Western analysis for p53 and p21 expression in NB-1643 cell extracts after exposure to Ad-VC at a moi of 10.

 
We also investigated whether heat-inactivated Ad-VC could induce p53 expression by a receptor-mediated pathway. Fig. 4Citation shows p53 and p21 expression in NB-1643 cells 24 h after exposure to both heat-inactivated (56°C for 1 h) and non-heat-inactivated virus. Inactivation of the virus was confirmed by a plaque-forming assay (15) . Results demonstrated that Ad-VC-mediated p53 induction was inhibited after heat inactivation of Ad-VC.

To further investigate the mechanism by which p53 is induced by adenoviral vector transduction, we carried out Northern analysis to determine whether induction was a result of an enhanced rate of transcription. RNA was isolated from NB-1643 cells prior to and after 24 h exposure to Ad-VC (moi of 10). Results demonstrated no increase in p53 RNA (data not shown), indicating that alterations in transcriptional regulation were not responsible for increasing p53 protein expression levels in the virally transduced cells.

We also investigated whether increased p53 protein stability was responsible for the induction observed after viral transduction in NB-1643 cells. Immunoprecipitation analysis using a p53 antibody (DO-1) with [35S]methionine-labeled cell extracts is shown in Fig. 5Citation . NB-1643 cells were transduced with Ad-VC for 24 h. Samples were obtained for immunoprecipitation immediately after a 1 h incubation with [35S]methionine and again after a further 2 h incubation in medium without isotope. The Ad-VC-transduced cells contained 2.3-fold more radiolabeled p53 protein immediately after a 1-h exposure to [35S]methionine than the nontransduced cells. However, after a further 2 h, the amount of [35S]p53 in both samples had reduced to ~50% (Fig. 5, B and C)Citation . This suggests that increased p53 expression after Ad-VC transduction is a result of increased p53 protein production and not of increased protein stability.



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  		Fig. 5. 35S-radiolabeled p53 immunoprecipitation of NB-1643 cell extracts with and without 24 h exposure to Ad-VC at a moi of 10. A, total cellular p53 after detection with p53 DO-1 antibody. B, 35S-labeled p53, as detected by autoradiography of the same membrane. C, quantitated amounts of 35S-labeled p53 protein as a histogram. Columns, means of two experiments; bars, span of the two individual points from which the mean was obtained. D, total cellular p53 after detection with p53 DO-1 antibody. E, 35S-labeled p53 generated after only a 15-min exposure to the isotope, as detected by autoradiography of the same membrane.

 
Because the half-life of p53 is only ~30 min (21) , the increase in 35S-labeled p53 protein observed after a 1 h labeling time in Ad-VC transduced cells could have been due to increased protein stability. Therefore, to minimize 35S-labeled p53 degradation, an immunoprecipitation was carried out after only a 15-min exposure to [35S]methionine. Results in Fig. 5ECitation demonstrate that the amount of 35S-labeled p53 increased 2-fold in Ad-VC-transduced cells after this short exposure time. Therefore, the overall increase in total cellular p53 after Ad-VC transduction appears to be due to an increased rate of translation and consequently an enhanced rate of p53 protein production.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Wild-type adenovirus infection has previously been shown to lead to increased levels of p53 protein in cells. This induction is mediated by the viral E1A protein (7) and inactivation by the E1B Mr 55,000 (8) and E4 Mr 34,000 (10) proteins. The Av1 adenoviral vectors used in this study and commonly in gene therapy express no E1A or E1B, and the p53 that we have observed to be induced after vector transduction is transcriptionally active.

Results shown in Fig. 1Citation demonstrate that an Av1 vector containing no transgene (Ad-VC) can induce p53 and p21 expression in NB-1643 neuroblastoma cells and IMR90 normal lung fibroblasts. This induction occurs at a rate slower than that after exposure of cells to DNA damage, suggesting a different induction mechanism. However, in both cases, the p53 expressed is transcriptionally active, as determined by subsequent induction of p21. Cell lines were also evaluated that expressed an attenuated DNA damage response because of either amplification of MDM2 or a mutated p53 gene. Ad-VC-mediated p53 induction was observed in cell lines containing a wtp53 gene, irrespective of MDM2 overexpression (Fig. 3)Citation . However, no induction was observed in Rh30 cells, which express a mutant p53 gene. These results also suggest that p53 induction is mediated by a separate pathway than that of DNA damage because, in NB-1691 MDM2-amplified cells, the DNA damage induction pathway is attenuated but the adenoviral vector pathway is functional. Additional cell lines with a wtp53 gene and an attenuated p53-DNA damage response need to be evaluated for Ad-VC mediated p53 induction before separate p53-induction pathways can be confirmed.

Adenovirus binds to a recently identified receptor also used by Coxsackie virus (22) . However, cellular internalization of adenoviral particles requires binding of the penton base viral coat protein to integrins (23) . Integrins have been found to be associated with a number of cell signaling molecules such as pp125FAK (focal adhesion kinase), which becomes phosphorylated and activated by integrin-ligand binding (24) . PI3K is a potential downstream substrate of focal adhesion kinase (25) , and it has been demonstrated that PI3K is activated after adenovirus-integrin binding and that this process is required for adenovirus internalization (20) . Because PI3K is involved in many signal transduction pathways (26) , we hypothesized that Ad-VC p53 induction could be mediated by PI3K activation. To test this hypothesis, we exposed NB-1643 cells exposed to wortmannin, an inhibitor of PI3K, together with Ad-VC at a moi of 10. However, wortmannin had no effect on viral-mediated p53 induction, demonstrating that PI3K signal transduction is not involved in this pathway of p53 induction (Fig. 4)Citation .

To further investigate whether a receptor-mediated pathway was responsible for the Ad-VC-mediated p53 induction, we evaluated whether heat-inactivated virus (56°C for 1 h) could induce p53 expression. Heat inactivation does not inhibit viral binding to its receptor but does prevent cellular expression of viral genes (27) , probably by inactivating the adenoviral protease required for escape from the endosome during endocytosis. However, heat inactivation of Ad-VC blocked the p53 induction (Fig. 4)Citation , indicating that expression of viral protein appears necessary to mediate p53 induction.

Northern analysis of RNA isolated from NB-1643 cells transduced with Ad-VC showed no increase in p53 RNA, demonstrating that enhanced p53 expression after exposure to adenoviral vectors was not a result of an enhanced transcription rate or increased stability of p53 RNA. Enhanced protein stability after viral transduction was also evaluated by immunoprecipitation analysis of 35S-labeled p53 protein. Results did not provide any evidence for increased p53 protein stability (Fig. 5)Citation ; however, an increased amount of radiolabeled-p53 protein was evident in cells exposed to Ad-VC, suggesting that there was enhanced p53 protein production and/or enhanced translation rate of p53 RNA (Fig. 5)Citation .

p53 protein negatively regulates its own RNA by binding to a stem-loop structure that forms in the 5'-untranslated region (28) . It is possible that adenoviral proteins may release this block on p53 translation or proteins may activate translation directly. Adenovirus infection is known to affect translation of cellular RNAs through alterations in the phosphorylation levels of eIF-4E and its inhibitor BP1, but this effect is mostly manifest at late stages of infection and is probably due to the activity of an E1A-encoded protein (29) . EIF-4E is responsible for cap-dependent mRNA translation, and modulation of its activity can have significant effect on the translation of cellular mRNAs. Whereas eIF-4E is likely the target of the translational effects we have observed, there are no obvious adenovirus proteins other than E1A that are candidates for modulating its activity.

The observation that replication-defective adenoviral vectors can initiate a functional p53 response in cells containing a wtp53 gene is important information for anyone using similar vectors for gene therapy. Because the vector itself can mediate changes in gene expression, it is very important that investigators always include appropriate negative controls in their studies, such that results obtained can be attributed to the specific transgene of interest and not to effects of the vector itself. Having said this, vector induction of wtp53 may be beneficial in certain types of gene therapy, particularly for treatment of cancer, because increases in p53 expression have been shown to mediate apoptosis and increase sensitivity to chemotherapeutic agents in model systems (30) .

The results presented here are also of importance to those interested in the biology and regulation of p53 expression as they describe identification of a novel mechanism leading to p53 induction which can enhance expression of wtp53, even in cells in which the DNA-damage induction pathway of p53 has been attenuated by the overexpression of MDM2.


   ACKNOWLEDGMENTS
 
We thank Dr. Janet Houghton for the Ad-VC vector and Queen Rodgers and Sean Nix for excellent technical assistance.


   FOOTNOTES
 
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.

1 This work was supported by NIH Grants CA 77541, CA 23099, and Cancer Center Core Grant P30-CA 21765 and by the American Lebanese Syrian Associated Charities. Back

2 Present address: Institute of Cancer Research, London, England SW3 6JB. Back

3 Present address: Genome Therapeutics Corporation, Waltham, MA 02453. Back

4 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38105. Phone: (901) 495-3833; Fax: (901) 521-1668; E-mail: linda.harris{at}stjude.org Back

5 The abbreviations used are: wtp53, wild-type p53; RSV, Rous sarcoma virus; moi, multiplicity of infection; HRP, horseradish peroxidase; PI3K, phosphoinositide-3-OH kinase. Back

Received 4/28/99. Accepted 7/19/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Levine A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323-331, 1997.[Medline]
  2. Teodoro J. G., Branton P. E. Regulation of apoptosis by viral gene products. J. Virol., 71: 1739-1746, 1997.[Medline]
  3. White E. Regulation of apoptosis by the transforming genes of the DNA tumor virus adenovirus. Proc. Soc. Exp. Biol. Med., 204: 30-39, 1993.[Medline]
  4. Oren M. Relationship of p53 to the control of apoptotic cell death. Semin. Cancer Biol., 5: 221-227, 1994.[Medline]
  5. Graham F. L., Abrahams P. J., Mulder C., Heijneker H. L., Warnaar S. O., de Vries F. A., van de Eb A. J. Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harbor Symp. Quant. Biol., 39: 637-650, 1975.
  6. Whyte P., Buchkovish K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. A. Association between an oncogene and an anti-oncogene: the adenovirus E1a proteins bind to the retinoblastoma gene product. Nature (Lond.), 334: 124-129, 1988.[Medline]
  7. Grand R. J. A., Grant M. L., Gallimore P. H. Enhanced expression of p53 in human cells infected with mutant adenoviruses. Virology, 203: 229-240, 1994.[Medline]
  8. Sarnow P., Ho Y., Williams J., Levine A. J. Adenovirus E1B-58K tumour antigen and SV40 large tumour antigen are physically associated with the same 54K cellular protein in transformed cells. Cell, 28: 387-394, 1982.[Medline]
  9. Flint S. J. Expression of adenoviral genetic information in productively infected cells. Biochim. Biophys. Acta, 651: 175-208, 1982.[Medline]
  10. Dobner T., Horikoshi N., Rubenwolf S., Shenk T. Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. Science (Washington DC), 272: 1470-1473, 1996.[Abstract]
  11. Roth J., Konig C., Weinzek S., Weigel S., Ristea S., Dobberstein M. Inactivation of p53 but not p73 by adenovirus type 5 E1B 55-kilodalton and E4 34-kilodalton oncoproteins. J. Virol., 72: 8510-8516, 1998.[Abstract/Free Full Text]
  12. Trapnell B. C. Adenoviral vectors for gene transfer. Adv. Drug Dev. Rev., 12: 185-199, 1993.
  13. Trapnell B. C., Gorziglia M. Gene therapy using adenoviral vectors. Curr. Opin. Biotechnol., 5: 617-625, 1994.[Medline]
  14. Wold W. S. M., Gooding L. R. Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology, 184: 1-8, 1991.[Medline]
  15. Becker T. C., Noel R. J., Coats W. S., Gomez-Foix A. M., Alam T., Gerard R. D., Newgard C. B. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol., 43: 161-189, 1994.
  16. Zhang W.-W., Koch P. E., Roth J. A. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. BioTechniques, 18: 444-447, 1995.[Medline]
  17. Houghton J. A., Harwood F. G., Tillman D. M. Thymineless death in colon carcinoma cells is mediated via Fas signaling. Proc. Natl. Acad. Sci. USA, 94: 8144-8149, 1997.[Abstract/Free Full Text]
  18. McPake C. R., Tillman D. M., Poquette C. A., George E. O., Houghton J. A., Harris L. C. Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status. Oncol. Res., 10: 235-244, 1998.[Medline]
  19. Momand J., Zambetti G. P., Olson D. C., George D., Levine A. J. The MDM2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69: 1237-1245, 1992.[Medline]
  20. Li E., Stupak D., Klemke R., Cheresh D. A., Nemerow G. R. Adenovirus endocytosis via {alpha}v integrins requires phosphoinositide-3-OH kinase. J. Virol., 72: 2055-2061, 1998.[Abstract/Free Full Text]
  21. Reid R. L., Lindholm P. F., Mireskandari A., Dittmer J., Brady J. N. Stabilization of wild-type p53 in human T-lymphocytes transformed by HTLV-I. Oncogene, 8: 3029-3036, 1993.[Medline]
  22. Bergelson J. M., Cunningham J. A., Droguett G., Kurt-Jones E. A., Krithivas A., Hong J. S., Horwitz M. S., Crowell R. L., Finberg R. W. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science (Washington DC), 275: 1320-1323, 1997.[Abstract/Free Full Text]
  23. Wickham T. J., Mathias P., Cheresh D. A., Nemerow G. R. Integrins {alpha}vß3 and {alpha}vß5 promote adenovirus internalization but not virus attachment. Cell, 73: 309-319, 1993.[Medline]
  24. Miyamota S., Akiyama S. K., Yamada K. M. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science (Washington DC), 267: 883-885, 1995.[Abstract/Free Full Text]
  25. Chen H-C., Guan J-L. Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA, 91: 10148-10152, 1994.[Abstract/Free Full Text]
  26. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinosi-tide. Nature (Lond.), 12: 673-676, 1997.
  27. Janik J. E., Huston M. M., Rose J. A. Locations of adenovirus genes required for the replication of adenovirus-associated virus. Proc. Natl. Acad. Sci. USA, 78: 1925-1929, 1981.[Abstract/Free Full Text]
  28. Mosner J., Mummenbrauer T., Bauer C., Sczakiel G., Grosse F., Deppert W. Negative feedback regulation of wild-type p53 biosynthesis. EMBO J., 14: 4442-4449, 1995.[Medline]
  29. Gingras A-C., Sonenberg N. Adenovirus infection inactivates the translational inhibitors 4E-BP1 and 4E-BP2. Virology, 237: 182-186, 1997.[Medline]
  30. Brown J. M., Wouters B. G. Apoptosis, p53 and tumor cell sensitivity to anticancer agents. Cancer Res., 59: 1391-1399, 1999.[Abstract/Free Full Text]



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