Adenoviral Bak Overexpression Mediates Caspase-Dependent Tumor Killing
Adenoviral Bak Overexpression Mediates Caspase-Dependent Tumor Killing
By Abujiang Pataer, MD, PhD, Bingliang Fang, MD, PhD, Jack A. Roth, MD, and Stephen G. Swisher, MD
Several recent clinical trials have demonstrated the feasibility of using adenovirally mediated gene transfer to treat lung and head and neck cancers.1,2 In these trials, adenovirally mediated overexpression of wild-type-p53 have produced clinical responses in heavily pretreated patients. It is clear, however, that not all patients respond to adenoviral-p53 and that in some patients who initially respond, the disease ultimately progresses and fails to respond to readministration of adenoviral-p53. There is a clinical need, therefore, to identify other candidate genes for use in treating cancer patients with adenoviral vectors. One promising group is the pro-apoptotic members of the Bcl-2 family (Bax, Bak), which have been shown to induce apoptosis following gene transfer via plasmid vectors in vivo.3,4
Background
Our initial attempts to develop an adenoviral vector with the pro-apoptotic Bak gene were complicated by the high level of toxicity induced in the 293 packaging cell line, and other authors have noted similar problems when trying to develop their own pro-apoptotic adenoviral vectors. Consequently, various strategies have been devised to overcome these problems. Shinoura and colleageus developed a packaging cell line expressing Crm-a, which, by blocking caspase activation, allowed production of an adenoviral Fas-L vector, while Okuyama opted for a Cre-mediated switching system.5,6 Tai and associates recently reported the development of a pro-apoptotic Bax vector whose expression in 293 cells and non-ovarian cancer cells was limited by the tumor-selective promoter DF3.7 Our own strategy was to use a binary adenoviral vector system in which Bak gene expression is transcriptionally controlled by the GT promoter and the Gal4/GV16 fusion protein. In this way, we have succeeded in producing an adenoviral vector in 293 packaging cells that can then be induced to express Bak in target tissues by coadministration with a Ad/GV16 vector producing the Gal4/GV16 fusion protein.8 This unique strategy allows high levels of Bak to be overexpressed in a variety of tissues both in vitro and in vivo.
Transduction Efficiency and Apoptosis
Using a binary adenoviral LacZ vector system (Ad/GT-LacZ + Ad/GV16), transduction efficiency (i.e., the titer required to achieve greater than 80% transduction) was determined for all cell lines and then used in subsequent experiments. Using these titers, high levels of Bak were induced when Ad/GT-Bak + Ad/GV16 was administered, but not when Ad/GT-LacZ or Ad/GFP was administered. Ad/GT-Bak induced high levels of apoptosis in vitro in human lung cancer cell lines as early as 24 hours after transduction. The mock-infected (PBS) or virus control-infected (Ad/GFP and Ad/GT-LacZ) cells demonstrated no apoptosis (1-3%), despite being infected with similar viral titers. The Ad/GT-Bak-infected cells, however, demonstrated 40-60% apoptosis 24-48 hours after infection.
To gain insight into the molecular effector pathway of Ad/Bak-induced apoptosis, we analysed whether caspase-9 and -3 were involved as downstream effectors in death signaling pathways mediating apoptosis in human lung cancer cells. Ad/Bak + Ad/GV16 resulted in activation of caspase-9 by cleaving it to 37 kDa subunit, and activation of caspase-3 by cleaving it to 17 kDa subunit, indicating that caspase-9 and -3 were activated during Ad/Bak-induced apoptosis. This apoptosis was confirmed by characteristic morphological changes and PARP cleavage. PARP and caspase-3, which are useful as indices of apoptosis, were only cleaved in cells that had been transduced with Ad/GT-Bak + Ad/GV16. MCF-7 cells were the only cells that failed to undergo apoptosis despite adequate Bak transduction. Interestingly, the levels of anti-apoptotic Bcl-2 family members did not appear to be greater in the MCF-7 cells than in the Bak sensitive cell lines, suggesting that these proteins were not the cause of the resistance in MCF-7 cells. However, MCF-7 cells differed from the other cell lines in lacking a functional caspase-3.9 Additionally, no PARP cleavage was noted in MCF-7 cells following Ad/GT-Bak infection, possibly because of the caspase-3 deficiency.
To investigate further whether Ad/GT-Bak-induced apoptosis and PARP cleavage were caspase dependent, we infected H1299 tumor cells with Ad/GT-Bak in the presence or absence of the caspase blocker z-DEVD-fmk. Bak-induced apoptosis was completely abrogated by caspase inhibition. Additionally, PARP cleavage was blocked by the addition of z-DEVD-fmk despite Bak overexpression. These results suggest that 1) MCF-7 cells may be resistant to Ad/GT-Bak killing because they are caspase deficient, and 2) Ad/GT-Bak-mediated tumor killing is caspase dependent.
Ad/GT-Bak and Tumor Size
We also evaluated the ability of Ad/GT-Bak to induce tumor regression in vivo in a subcutaneous nu/nu tumor mouse model. The Ad/GT-Bak-injected tumors were significantly smaller than both the saline control and virus-control infected tumors (P < 0.05). In addition, these observations held true in both p53 wild-type (A549) and p53 null cells (H1299), suggesting that the mechanism was p53 independent. To determine if this effect was caused by the induction of apoptosis, we isolated tumors 12 hours after the initial viral treatment, sectioned them, and then subjected them to histology and TUNEL assays. Compared with the tumors of control mice, Ad/GT-Bak-injected tumors were smaller, demonstrated more of the cellular debris and morphological changes associated with apoptosis, and demonstrated increased TUNEL staining. In contrast, treatment with Ad/GT-LacZ or Ad/GFP yielded no histologically detectable apoptotic responses. Imprtantly, intratumoral injection of Ad/GT-Bak caused no significant systemic toxicity. Together, these results demonstrate the potential antitumor effect of Ad/GT-Bak when directly injected into tumors.
Conclusion
Our studies also suggest that this process is p53 independent since Bak-induced apoptosis occurred equally well in p53 wild-type, null, or mutant cells. This observation may be explained by the fact that the Bcl-2 family members function downstream from p53 in the apoptotic cascade. Indeed, we have observed that Ad-p53 appears to induce apoptosis in part by initial upregulation of Bak and Bax followed by apoptosis induction, perhaps by cytochrome c release and caspase activation.10 These observations are important because if Bak functions downstream from p53, then the clinical use of the Bak vector may be independent of p53. If so, then treatment of p53 mutant or resistant cancer cells with adenoviral Bak would be possible. It has also been observed that the gene transfer of pro-apoptotic Bax via lipid transfection is more cytotoxic than p53 gene transfer. However, any increased apoptotic potential of the pro-apoptotic Bcl-2 family members may come at a price, since we have observed that Ad/GT-Bak appears to be less selective than Ad/p53 in killing normal human bronchial epithelial cells.
We and others are seeking solutions to this problem of toxicity to normal tissue. For example, Tai et al have proposed using the selective promoter DF3 with a Bax vector and we have evaluated the use of the telomerase promoter.7 The disadvantage to the selective promoter strategy, however, is that only tumor tissues that are permissive to the DF3 or telomerase promoter would be treatable. Alternatively, the strategy of directly injecting a binary adenoviral vector into a tumor may, by localizing vector delivery, allow most tumor tissues to be transduced while minimizing the toxicity to normal tissue. The fact that we noted no systemic toxicity in our nude mouse model following direct intratumoral injection of Ad/GT-Bak suggests that localized delivery may indeed be a reasonable clinical strategy. (Dr. Pataer is Postdoctoral Fellow, Dr. Fang is Assistant Professor, Dr. Roth is Professor and Chairman, and Dr. Swisher is Assistant Professor and Assistant Surgeon, Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX.)
References
1. Swisher SG, Roth JA, Nemunaitis J, et al. Adenoviral-mediated p53 gene transfer in advanced non-small cell lung cancer. J Natl Cancer Inst 1999;91:763-771.
2. Clayman GL, El-Naggar AK, Lippman SM, et al. Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J Clin Oncol 1998;16:2221-2232.
3. Coll JL, Negoescu A, Louis N, et al. Antitumor activity of bax and p53 naked gene transfer in lung cancer: In vitro and in vivo analysis. Hum Gene Ther 1998;20:2063-2074.
4. Orth K, Dixit VM. Bik and Bak induce apoptosis downstream of CrmA but upstream of inhibitor of apoptosis. J Biol Chem 1997;272:8841-8844.
5. Okuyama T, Fujino M, Li XK, et al. Efficient Fas-ligand gene expression in rodent liver after intravenous injection of a recombinant adenovirus by the use of a Cre-mediated switching system. Gene Ther 1998;5:1047-1053.
6. Shinoura N, Ohashi M, Yoshida Y, et al. Construction, propagation, and titer estimation of recombinant adenoviruses carrying proapoptotic genes. Hum Gene Ther 1998;9:2683-2689.
7. Tai YT, Strobel T, Kufe D, et al. In vivo cytotoxicity of ovarian cancer cells through tumor-selective expression of the BAX gene. Cancer Res 1999;59:2121-2126.
8. Fang B, Ji L, Bouvet M, et al. Evaluation of GAL4/TATA in vivo: Induction of transgene expression by adenovirally mediated gene codelivery. J Biol Chem 1998;273:4972-4975.
9. Kurokawa H, Nishio K, Fukumoto H, et al. Alteration of caspase-3 (CPP32/Yama/apopain) in wild-type MCF-7, breast cancer cells. Oncol Rep 1999;6:33-37.
10. Pearson AS, Spitz FR, Swisher SG, et al. Upregulation of the proapoptotic mediators bax and bak following adenovirus-mediated p53 gene transfer in lung cancer cells. Clin Cancer Res 2000;6:887-890.
Crm-a:
a. activates caspase.
b. blocks caspase activation.
c. activates superoxide dismutase.
d. blocks superoxide dismutase activation.
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