Background
A number of clinical trials for cancer therapy with replication-competent viruses have been conducted and some of the agents are approved in China, Unites States and Europe [
1,
2]. Adenoviruses (Ad) are one of the agents extensively investigated and are easy to be genetically manipulated to produce replication-restricted types for human tumors. There are mainly 2 structural categories that make preferential replications in tumors, Ad defective of a region that inhibits viral replications in non-tumorous cells such as the E1B 55 kDa-defective type [
3] and Ad of which the E1A region is activated with a transcriptional regulatory unit of a gene which is preferentially up-regulated in human tumors. Prediction of Ad-mediated cytotoxicity is important for selecting candidate patients who are suitable for the virotherapy in a clinical setting but such a predictive biomarker for the cytotoxicity remains uncharacterized.
Efficacy of the viral replication-mediated cell death can be influenced by Ad infectivity and also by a transcriptional activity of an exogenous promoter region to activate the E1A region in the second type. Nevertheless, few reports extensively analyzed correlation between the Ad-mediated cytotoxicity and the infectivity or the E1A-activating capacity. On the other hand, further understanding of Ad biology enabled us to produce modified Ad of which the infectivity was changed by replacing the fiber-knob region since the region mediated Ad binding to the cellular receptors [
4]. Ad use different receptor molecules, depending on the subtypes. Consequently, substituting the fiber-knob region can convert the infectivity based on the Ad subtypes. Conventional Ad vector belongs to type 5 (Ad5) and uses coxsachie adenovirus receptor (CAR) as the main cellular receptor and intergrin αvβ3 and αvβ5 as the axillar receptor, whereas type 35 Ad (Ad35) vector uses CD46 as the main receptor [
5]. Ad5 bearing the Ad35-derived fiber-knob structure (AdF35) therefore infected CD46-positive cells irrespective of CAR expression [
6,
7]. The expression levels of CAR molecules in human tumors were variable and often down-regulated, rendering replication-competent Ad5 less cytotoxic to human tumors [
8]. In contrast, CD46 was ubiquitously expressed in human cells and the expression was rather up-regulated in a number of human tumors [
9]. AdF35 can therefore infect human tumors better than Ad5 [
10] and consequently produced greater cytotoxicity [
11].
Cytotoxic activities of the replication-competent Ad of which the E1A is regulated by an exogenous regulatory region can also be attributable to transcriptional activities of the region in target cells. We and others previously showed that a 5′ untranslated region of
midkine (MK) [
12],
survivin (Sur) [
13] or
cyclooxygenase-2 (COX-2) gene [
14], all of which were up-regulated in the expression in a number of human tumors, activated a reporter gene in human tumors but much less in human normal cells. Replication-competent Ad powered by the promoter region in fact produced preferential cytotoxicity in various type of human tumors with little damages in non-transformed cells [
15‐
17]. Replacement of the fiber-knob region with the Ad35-derived one can widen the target tumor scopes and furthermore produce better cytotoxicity [
18]. In a clinical setting, a possible biomarker to predict the efficacy of these Ad is desirable to narrow down candidate patients. We therefore tested the cytotoxicity of replication-competent Ad5 and AdF35 bearing the same transcriptional regulatory region in 3 kinds of human tumors which include 4 pancreatic, 9 esophageal carcinoma and 5 mesothelioma cell lines, and examined whether Ad infectivity and the transactivation activity could be a predictive marker. We also examined a possible linkage between the
p53 genotype and the cytotoxicity with the esophageal carcinoma.
Methods
Cells
Human pancreatic carcinoma, PANC-1 (TKG 0606,
p53 genotype: mutated), AsPC-1 (JCRB1454, null), MIA-PaCa-2 (TKG 0227, mutated) and BxPC-3 (JCRB1448, mutated) cells, and human esophageal carcinoma, TE-1 (TGK 0252, mutated at codon 272 Val to Met), TE-2 (TGK 0253, wild-type), TE-10 (TKG 0261, mutated at codon 242 Cys to Tyr), TE-11 (TKG 0262, wild-type), YES-2 (mutated at codon 236 Tyr to Asn) [
19], YES-4 (wild-type) [
20], YES-5 (mutated at codon 280 Arg to Gly) [
20], YES-6 (wild-type) [
20] and T.Tn (JCRB 0261, mutated at codon 214 His to Arg and 258 Glu to stop) cells were from Cell Resource Center for Biomedical Research (TKG number; Sendai, Japan), National Institutes of Biomedical Innovation, Health and Nutrition (JCRB number; Tokyo, Japan) or Dr. Yutaka Shimada (YES-2, YES-4, YES-5 and YES-6; Kyoto University, Kyoto, Japan). HEK293 cells (CRL-1573) and human mesothelioma, NCI-H2452 (CRL-5946, wild-type but truncated p53 protein), NCI-H2052 (CRL-5915, wild-type), NCI-H226 (CRL-5826, wild-type), NCI-H28 (CRL-5820, wild-type) and MSTO-211H (CRL-2081, wild-type) cells, were from ATCC (CRL number; Manassas, VA, USA). All the cells were cultured with RPMI 1640 supplemented with 10% fetal calf serum.
Construction of ad
Replication-incompetent Ad5 expressing the
β green fluorescence protein gene (GFP) (U55762) powered by cytomegalovirus promoter (Ad5/GFP) were prepared with Adeno-X expression system (Takara, Shiga, Japan), which included ligation of transgene-harboring pShuttle 2 and Adeno-X vectors followed by transfection into HEK293 cells. AdF35, bearing the above transgene (AdF35/GFP or AdF35/LacZ), were produced with the Adeno-X vector of which the corresponding genomic fragment (AY271307 at 30827–33609) was replaced with that of the Ad35 DNA (Avior Therapeutic, Seattle, WA, USA). These replication-incompetent Ad5 and AdF35 vectors used the same cytomegalovirus promoter (BK000394) to activate the respective genes. Replication-competent Ad5 or AdF35 in which the
E1 gene was activated by an exogenous regulatory element, Ad5/Sur, Ad5/MK, Ad5/COX-2, AdF35/Sur, AdF35/MK and AdF35/COX-2, were prepared by replacing the authentic E1 promoter region with 5′ upstream regulatory sequences of the
MK (0.6 kb, D10604) [
12] the
Sur (0.5 kb, U75285) [
13], or
COX-2 (0.3 kb, U04636) gene [
14]. Ad were purified with an Adeno-X virus purification kit (BD Biosciences, San Jose, CA, USA) and the numbers of virus particles (vp) per ml was estimated with the formula, absorbance at 260 nm of purified Ad in the presence of 0.1% sodium dodecyl sulfate × 1.1 × 10
12.
Cytotoxicity of ad
Cells (5 × 103/well) were seeded in 96-well plates and were cultured for 5 days with different amounts of Ad (vp/cell). Cell viability was determined with a cell proliferation colorimetric WST kit (Wako, Osaka, Japan). The amount of formazan produced was determined with the absorbance at 450 nm and the relative viability was calculated based on the absorbance without any treatments. Half maximal inhibitory concentration (IC50) was estimated with CalcuSyn software (Biosoft, Cambridge, UK).
Cells were infected with Ad5/GFP or AdF35/GFP at 30 multiplicity of infection (MOI) for 30 min and were washed to remove Ad. Infected cells were cultured for 2 days and then analyzed for percentages of GFP-positive cells with FACSCalibur (BD Biosciences) and CellQuest software (BD Biosciences). Cells of which fluorescence was greater than the brightest 5% of uninfected cells were judged as positively stained.
Expression of ad receptor molecules
Cells were stained with either anti-CAR antibody (Ab) (#05–644, Upstate, Charlottesville, VA, USA) followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG Ab or with FITC-conjugated anti-human CD46 Ab (#555949, BD Pharmingen, San Jose, CA, USA). They were then analyzed for their fluorescence intensity with FACSCalibur and CellQuest software. Mean fluorescence intensity of the staining profiles was expressed as an arbitrary FL1 unit after standardizing intensity by the second antibody, FITC-conjugated or isotype-matched control Ab as 10 in the unit.
Transcriptional activity
Genomic fragments of a 5′-transcriptional regulatory region of the 0.6 kb
MK [
12], the 0.5 kb
Sur [
13], or the 0.3 kb
COX-2 [
14] gene were cloned into pGL-2 basic vector (Promega, Madison, WI, USA) that contained the
firefly luciferase gene. Plasmid DNA containing the respective genomic fragments, pGL-control vector (Promega) harboring the SV40 T antigen promoter-linked
firefly luciferase gene, or pGL-basic vector without any transcriptional regulatory regions (Promega), and a control vector, the
renilla luciferase gene fused with the
herpes simplex virus-thymidine kinase gene promoter (pRL-TK, Promega), at a molar ratio of 10:1, was transfected into tumors with a lipofectin reagent (Life Technologies, Gaithersburg, MD, USA). Cell lysate on day 2 was assayed for the luciferase activity with the dual luciferase reporter assay (Promega). The firefly luciferase activity was standardized with the amounts of luminescence produced by renilla luciferase and the relative activity was expressed as a percentage of the SV40 T antigen promoter-mediated activity.
Western blot analysis
Lysate of cells treated with Ad was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was transferred to a nylon filter and was hybridized with Ab against γ-H2A histone family member X (γ-H2AX) (#613401, BioLegend, San Diego, CA, USA), p53 (DO-10 MS-187-P, Thermo Fisher Scientific, Fremont, CA, USA), p21 (#2947, Cell Signaling, Danvers, MA, USA) or β-actin (#4970, Cell Signaling) as a control. The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK).
Discussion
We investigated in the present study a possible correlation between cytotoxicity produced by replication-competent Ad and the infectivity/gene expression or a transcriptional activity of an exogenous regulatory region that was used to activate E1 region genes. We produced AdF35 which differed only in the fiber-knob region and compared with the prototype Ad5 in the cytotoxicity and the infectivity. The present study demonstrated that replication-competent AdF35 produced greater cytotoxicity than the prototype Ad5 bearing the same regulatory region, but the cytotoxicity irrespective of the Ad types was not correlated with the infectivity/gene expression or transcriptional activity of the region used for activation of E1 genes. Nevertheless, we demonstrated that the p53 genotype differentiated the sensitivity of esophageal carcinoma to the Ad-mediated cytotoxicity with greater cytotoxicity in p53-mutated cells than in p53 wild-type cells.
A biomarker to predict an oncolytic ability of replication-competent Ad is important in the clinical applications. The biomarker is useful to select a patient who responds to the Ad-mediate cancer therapy and to exclude a patient who suffers from severe adverse effects caused by the gene medicine. Improved cytotoxicity of AdF35 in comparison with the corresponding Ad5 could be attributable to the enhanced infectivity. Expression of CAR molecules, the major cellular receptor of Ad5, was often down-regulated in human tumors and in fact the present study showed that the CAR expression levels in 3 kinds of human tumors were lower than that of HEK293 cells. In contrast, the level of CD46 molecules, the major receptor for Ad35, did not decrease in the tumors and was rather higher than that in HEK293 cells. The fiber-knob region of Ad5 and Ad35 is responsible for binding with CAR and CD46 molecules, respectively, and replacement of the Ad5-derived region with the Ad35 region ablated the CAR binding ability and enabled AdF35 bind to CD46 molecules. Nevertheless, cytotoxicity of replication-competent Ad5 or AdF35 was not directly correlated with infectivity/gene expression of Ad5 or AdF35 vector irrespective of the transcriptional regions used. These data suggest that expression of subsidiary Ad receptors such as integrin αvβ3 and αvβ5 can also be pivotal for Ad infectivity/gene expression. The present study demonstrated that GFP-positive percentages produced by Ad5 or AdF35 vector were unrelated with CAR and CD46 levels, respectively. Ad-mediated gene expression is regulated at various steps and the infection process can also be influenced by a threshold of the receptor expression. The GFP expression was thereby not directly or linearly associated with the major receptor expression levels. Contribution of the major receptors to the infectivity can be limited in particular in cells with CAR-low or CD46-low expression. Lyle et al. in fact demonstrated that integrin αvβ5 worked as the primary receptor in CAR-negative cells [
23]. Previous studies also suggested that infectivity of Ad5 or Ad5 bearing type 11-derived fiber-knob region, which used CD46 molecules as the major receptor, was not directly correlated with the cytotoxicity although the studies did not analyzed statistically [
24]. Increased CAR expression augmented Ad5 infectivity and the Ad5-mediated cytotoxicity [
25,
26], but correlation between the CAR expression and Ad5 infectivity was not extensively investigated. In contrast, the present study statistically demonstrated that increased Ad infectivity/gene expression was not associated with the Ad replication-mediated cytotoxicity.
We also analyzed the E1-activating ability of Ad in the infected cells since E1A protein or the transcript levels were linked with the cytotoxic activity of the Ad [
24,
27]. The present study showed that the transcriptional activity of respective regulatory regions varied depending on target cells and the region integrated in Ad. The activities of MK and COX-2 were constantly greater than that of SV40 T antigen, whereas the Sur activity was variable in comparison with that of SV40 T antigen. The variability of Sur activities in the tumors tested could be partly attributable to preferential expression of the
Sur gene at G2/M phase [
28]. Nevertheless, the present study demonstrated that the E1A-activation ability was not directly correlated with the cytotoxicity except one case, AdF35/Sur-mediated cytotoxicity and Sur activity in pancreatic carcinoma. The previous studies which analyzed a possible linkage between E1A expression and the cytotoxicity did not analyze statistical significance [
24,
27], but the present study was to our knowledge the first report to demonstrate no significant association between them. These data consequently suggest that a cellular factor play an important role in the Ad-mediated cytotoxicity. A mechanism of Ad replication-induced cell death is complex and the cell death pathways might be different among the cells tested.
We examined a correlation between the
p53 genotype and the cytotoxic activity with esophageal carcinoma and demonstrated that cells with wild-type
p53 gene were resistant to Ad replication-induced cytotoxicity compared with those with mutant
p53 in particular with the AdF35 vectors. Previous studies showed that transduction with the wild-type
p53 enhanced cytotoxicity produced by replication-competent Ad [
29,
30] and that the Ad-mediated cytotoxicity, which was further augmented by co-expressed p53, was independent of the p53 status of target cells [
30,
31]. Expression of E1A accompanied by the viral replications enhanced expression of p53 and the phosphorylation, which contributed to augmentation of cell death. In contrast, the E1A-induced phosphorylation of mutated p53, functioned as a dominant-negative form, increased the resistance to cell death, which consequently augmented viral replications and production of viral progenies. The differential susceptibility of replication-competent Ad in terms of the
p53 genotype can be attributable to how infected cells were subjected to death and to how much viral progenies were produced through preventing premature cell death. A number of factors were involved in a balance between survival and death signals, such as differential activities between apoptotic and anti-apoptotic pathways, and autophagy and anti-autophagy pathways, as well as cellular components that influence viral progeny production. Further investigations, for example, a treatment with siRNA for p53, are required to clarify a possible role of p53 in the Ad-mediated cytotoxicity.
Functional significance of p21, one of the p53 down-stream molecules, in the Ad-mediated cytotoxicity was controversial. Flak et al. showed that cells with inducible p21 were susceptible to replication-competent Ad [
21], whereas Höri et al. demonstrated that a chemical agent to augment p21 expression decreased the cytotoxicity [
22]. We treated cells with cisplatin, a representative agent to induce DNA damages, and examined p21 expression. Cells infected with replication-competent Ad were not used since they were difficult to be standardized for the DNA damages during viral replications. Increased p21 expression can inhibit cell cycle progression at G1 phase and consequently decreases viral replications. On the other hand, p21 is inhibitory to cell death, which makes cells alive and productive of viral particles. A functional role of p21 in viral replications and the cytotoxicity is thus divalent and can be differentially influenced by properties of the infected cells. The current study showed that decreased p21 expression after DNA damages was associated with increased susceptibility to Ad-mediated cytotoxicity and indicated that down-regulation of p21 facilitated cell cycle progression to make cells competent for viral replications and favored cell death. Biological significance of the down-regulated p21 in terms of the cytotoxicity needs further studies since Ad-mediated cytotoxicity is influenced not only by viral replications but susceptibility to cell death mechanisms. We noticed that the mutated
p53 esophageal carcinoma tended to decreased p21 expression, and consequently mutated
p53 genotype and decreased p21 levels could be relevant to each other regarding the Ad-mediated cytotoxicity. In addition, responses of p53 and p21 to cisplatin in esophageal carcinoma were different from typical damage responses, which suggested that the p53 pathways were impaired. We however found that cisplatin-treated cells induced cleavage of PARP and caspase-3 in all the
p53 wild-type esophageal carcinoma (Additional file
8: Figure S8), indicating that apoptosis was induced by cisplatin. We also showed that association of the cytotoxicity with the
p53 genotype or with the p21 responses was greater with AdF35 than with Ad5 vectors, but the mechanism behind this vector difference was currently unknown.
The present study suggested the
p53 genotype as a potential biomarker to predict the efficacy but this outcome needs to be confirmed with clinical specimens. Moreover, expression of cellular proteins necessary for Ad replications such as nuclear factor-1 and production of type I interferon followed by Ad infection are also issues to be examined since these factors also influence susceptibility of target cells to Ad-mediated cell death [
32‐
34]. Prediction of Ad-mediated cytotoxicity is important from the standpoint of the possible clinical application, and further investigations are required to establish such predictive markers because genetic and epigenetic alterations in target cells are involved in the Ad-mediated cytotoxicity.