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p14^ARF Modulates the Cytolytic Effect of ONYX-015 in Mesothelioma Cells with Wild-type p53

Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan [C-T. Y.]; Thoracic Oncology Laboratory, UCSF Cancer Center, University of California, San Francisco, California 94115 [C-T. Y., L. Y., K. U., F. M., D. M. J.]; and Molecular Urology Laboratory, Mt. Zion Hospital, University of California, San Francisco, California 94115 [C-C. Y.]

	ABSTRACT

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ONYX-015 has been reported to kill selectively tumor cells lacking functional p53. Genetic alterations of INK4a/ARF locus, which is a predominant event in malignant pleural mesothelioma, may result in loss of p14^ARF and subsequent disruption of p53 pathway in cancer cells. In the present study, ONYX-015 was able to kill three mesothelioma cell lines (H28, H513, and 211H) with wild-type p53 but lacking p14^ARF. In contrast, MS-1 mesothelioma cells, which expressed both p53 and p14^ARF, were resistant to ONYX-015. Introducing p14^ARF gene into the H28 cell, a mesothelioma cell without p14^ARF expression, significantly increased the resistance of this cell line to the cytolytic effect of ONYX-015. Our results suggest that human mesotheliomas with wild-type p53 yet lacking p14^ARF are potential candidates for ONYX-015 therapy.

	Introduction

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MPM² is an asbestos-related malignancy characterized by rapidly progressive and diffusely local growth, late metastases, and poor prognosis. Approximately 3,000 patients are diagnosed with MPM in the United States annually. The incidence of this disease worldwide is rising and is expected to peak in the next 2 decades (1) . Chemotherapy or radiation therapy, alone or in combination, is not effective, and the majority of patients with MPM die within 2 years regardless of treatment (1) . Considering the uniformly fatal outcome and lack of effective treatment, the development of novel treatment strategies for MPM given the lack of response with conventional therapies is therefore needed. Replicating oncolytic virus is a promising new modality for cancer treatment. The strategy of this therapy is to develop viruses capable of selectively infecting malignant tumors which can spread and destroy malignant tumors without deleterious effects in normal tissues. The key to the development of such viruses is the identification of viral genes whose deletion or modification enables tumor-specific cell killing. ONYX-015, a conditional replication-competent adenovirus lacking E1b 55 kDa gene, is a promising agent based on this strategy. ONYX-015 contains an 827-bp deletion in the E1b region and a point mutation at codon 2022 that generates a stop codon. This genetic design takes advantage of the fact that adenovirus E1b 55k binds and thus inactivates wild-type p53 protein. This binding/inactivation is essential to virus replication. ONYX-015 cannot replicate in normal cells but can replicate in tumor cells lacking functional p53 (2) . ONYX-015 has been shown to kill cervical, colon, pancreatic carcinoma cells, glioma cells, and non-small cell lung cancer cells lacking functional p53 with comparable efficiency to wild-type adenovirus (2 , 3) . In mesothelioma, unlike other adult malignancies, genetic alterations in p53 are rare (4) . Homozygous deletions of INK4a/ARF locus, however, have been shown to be the predominant events which occur at a frequency of >70% in this malignancy (5 , 6) . The INK4a/ARF locus (7) on human chromosome 9p21 plays an important role in both the pRB and p53 tumor suppressor pathways by encoding two distinct proteins translated from alternatively spliced mRNAs. p16^INK4a has been biochemically characterized as a protein that specifically binds to and inhibits cyclin-dependent kinase-4/6. Thus, p16^INK4a regulates pRB phosphorylation and induces cell cycle arrest in G₁ phase (8) . p14^ARF plays a role as a negative regulator of MDM2, interfering with MDM2-mediated shuttling and degradation of p53 (9, 10, 11) . A single mutational event at the INK4a/ARF locus therefore has the potential to disrupt both pRB and p53 tumor suppressor pathways. In addition, the p14^ARF gene promoter is a CpG island that can be silenced by DNA methylation (12) . These genetic alterations may result in a loss of negative regulation of MDM2. Because MDM2 has functional similarity to the adenoviral E1B 55K protein, lack of p14^ARF may thus prevent a normal p53 response to viral infection, thereby allowing ONYX-015 replication. In the present study, we showed that ONYX-015 killed effectively mesothelioma cells with normal p53 gene but lacking p14^ARF expression. Introducing the p14^ARF gene into human mesothelioma cells lacking p14^ARF expression by use of an adenoviral vector significantly increased the resistance of cells to the CPE of ONYX-015. This study suggests that ONYX-015 may be an effective treatment for tumors retaining wild-type p53 but lacking p14^ARF, such as MPM.

	Materials and Methods

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Cell Lines and Cell Culture.
H28 (ATCC CRL-5820), H513 (ATCC CRL-5830), and MSTO-211H (ATCC CRL-2081, 211H) mesothelioma cells and Met5A (ATCC CRL-9444) transformed mesothelium cells were obtained from American Type Culture Collection. MS-1 mesothelioma cell line has been described previously (13) . All were cultured in RPMI 1640 complete media containing 10% FCS.

CPE Assay.
ONYX-015 and WTD were supplied by ONYX Pharmaceuticals (Richmond, CA). WTD is identical to ONYX-015 except in the E1B, 55-kDa gene region where the original, wild-type sequence is present. Cells (1 x 10⁵ cells/well) were plated in replicate 6-well plates, incubated overnight at 37°C, and then infected with ONYX-015 or WTD at increasing MOI, i.e., 0, 0.01, 0.1, and 1 plaque-forming unit per cell, in RPMI 1640 containing 2% FCS for 4 h at 37°C. Then cells were incubated in complete medium containing 10% FCS, and plates were monitored daily for CPE. The assay was terminated when essentially total cytolysis was observed in cultures infected with WTD at an MOI of 0.1. The plates were then stained with crystal violet (0.5% in 20% methano; Sigma Chemical Co., St. Louis, MO) for analysis.

Adenoviral Vectors.
The adenoviral vectors for gene expression studies were constructed by using the AdEasy System (14) . The p14^ARF and p16^INK4A cDNA were kindly provided by Dr. K. Vousden (National Cancer Institute, Frederick, MD) and Dr. D. Beach (Institute of Child Health, London, England, United Kingdom). Briefly, the gene of interest was first cloned into a shuttle vector, pAdTrack-CMV, which contained CMV promoters and a GFP gene. The resultant plasmid was subsequently cotransformed into Escherichia coli BJ5183 cells with an adenoviral backbone plasmid, pAdEasy-1. p14^ARF and p16^INK4A recombinant adenoviruses (Adp14 and Adp16, respectively) were then generated in 293 cells (E1-transformed human embryonic kidney cells) and identified by PCR analysis of the DNA samples prepared from the cell culture supernatant. The recombinant adenovirus, AdCtrl, which carries a GFP gene regulated by the CMV promoter, was constructed with pAdTrack and pAdEasy-1 and used as a control in these experiments.

Immunoblotting.
Cells (1 x 106) were plated in 10-cm dishes and incubated for 4 h at 37°C. Cells were then mock infected or infected with either AdCtrl or Adp14 (MOI of 50 each) and incubated for 72 h. Cell samples were washed twice with PBS, scraped off the plates, and lysed in cell lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS]. Whole cell lysates were boiled, and the protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Balanced amounts of cell proteins (20–40 µg) were boiled again after the addition of mercaptoethanol and bromphenol blue and fractionated by SDS-PAGE in 4–20% linear gradient acrylamide gel (Ready-Gel; Bio-Rad). After transferring the proteins onto Immobilon-P (Millipore, Bedford, MA), the membranes were blocked in 5% nonfat milk powder, 0.2% Tween 20 in TBS overnight at 4°C, and incubated with the primary antibody for 1 h at room temperature. Membranes were then washed in Tween 20 in TBS for 5 min three times. Primary antibodies for p14^ARF (C-18), p21^WAF1 (C-19), and p53 (DO-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Depending on the experiment, goat antimouse horseradish peroxidase and donkey antigoat horseradish peroxidase were used as secondary antibodies (Santa Cruz Biotechnology). Antibody binding was visualized using Chemiluminescence Luminol reagents (Santa Cruz Biotechnology).

Cytotoxicity Assays.
Cells were plated in triplicate on 96-well culture plates (500 cells/well) and incubated overnight at 37°C. Cells were then exposed to varying concentrations of AdCtrl, Adp14, or Adp16 and incubated for 7 days at 37°C. A colorimetric assay was performed as described previously (15) . Briefly, cells were fixed in 10% trichloroacetic acid for 1 h, washed five times with water, and allowed to air dry. Cells were then stained for 30 min with 0.4% sulforhodamine B (Sigma Chemical Co.), dissolved in 1% acetic acid, and rinsed five times with 1% acetic acid to remove unbound dye. Bounded dye was then solubilized with 10 mM unbuffered Tris-base (pH 10.5) for 5 min. Visual absorbance (A595) was obtained using a kinetic microplate reader (Vmax; Molecular Devices, Sunnyvale, CA), which was used as a measure of cell number. The IC₅₀ (dose which inhibited cell growth by 50%) was calculated assuming the survival rate of uninfected cells to be 100%. The relative differences in IC₅₀s were calculated by dividing the IC₅₀ of cells infected with Adp14 or Adp16 by the IC₅₀ of cells infected with AdCtrl for each cell line.

Measurement of Cell Viability.
H28 cells (1 x 10⁵ cells/well) were seeded into 6-well tissue culture plates, incubated overnight at 37°C, and then mock infected or infected with either WTD or ONYX-015 (MOI of 1 each) in combination with Adp14 or AdCtrl (MOI of 10 each) in RPMI 1640 containing 2% FCS for 4 h at 37°C. Cells were then incubated in complete medium containing 10% FCS until complete cell lysis was observed in cultures treated with both WTD and AdCtrl. Then cells in the remaining wells were collected by trypsinization and suspended in PBS. An equal volume of 0.4% trypan blue (Sigma Chemical Co.) was added to the cell suspension. Viable cells were then determined by direct microscopic counting. All counts were done on triplicate samples.

Statistical Method.
Results are expressed as means ± SD. Statistical comparisons were made by two-sided t test. A value of P < 0.05 was accepted as significant.

	Results

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CPE of ONYX-015 in Human Mesothelioma Cells.
The four mesothelioma cell lines, H28, H513, 211H, and MS-1, used in the present study were examined previously by single-strand conformation polymorphism, and no mutations in exons 5–8 of the p53 gene were detected (data not shown). ONYX-015, however, can effectively lyse H28 at MOI of 0.1, and both H513 and 211H were lysed at MOI of 1 within 10 days. Only MS-1 was resistant to ONYX-015 up to MOI of 1 (Fig. 1) .

View larger version (78K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. CPE of ONYX-015 on mesothelioma cells. ONYX-015 (015) lysed H28 cells completely at MOI of 0.01; however, MS-1 cells were resistant to ONYX-015 up to MOI of 1. Wild-type adenovirus (WT) was used as a positive control for CPE. The cell lines used are shown to the right, the infectious agents are shown to the left, and the titers are above.

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. A, immunoblot analysis of p53 and p14ARF in mesothelioma cells. In mesothelioma cell line H28, endogenous p14ARF expression was absent, whereas substantial expression of p14ARF was detected by immunoblotting after infection with Adp14, a p14ARF-expressing adenoviral vector. Infection with Adp14 also markedly increased p53 expression in H28 cells. Endogenous p53 and p14ARF expressions were detected in MS-1 mesothelioma cells. The Met5A transformed mesothelium cell line was used as a positive control for the expressions of both p14ARF and p53. The cell lines used are shown above their corresponding lanes, and the antibodies are shown to the left. B, immunoblot analysis of p14ARF, p21WAF1, and p53 in MS-1 cells at 3, 6, 9, 12, and 24 h after ONYX-015 infection. The time periods after the infection are shown above their corresponding lanes, and the antibodies are shown to the right.

Cytostatic Effect of Adenovirus-mediated p14^ARF or p16^INK4A Expression in H28 and MS-1 Mesothelioma Cells.
Both p14^ARF and p16^INK4A function as the tumor suppressors, and they have been targeted for gene-replacement strategies for cancer therapy (7 , 8 , 15 , 17 , 18) . We additionally studied the cytostatic effect of gene replacement with p14^ARF or p16^INK4A in H28, the cell lacking expression of both the p14^ARF and p16^INK4A proteins. We also studied MS-1, a cell line with p14^ARF expression but deficient in p16^INK4A, by examining the ratios of the IC₅₀s after Adp14 or Adp16 infection relative to the IC₅₀s of AdCtrl. Although the cytostatic effect of Adp16 varied between these two cell lines, the IC₅₀s of Adp16 in both cells were 10-fold (8) lower than that of AdCtrl. The IC₅₀ of Adp14 for H28 cells was 8-fold lower relative to that of AdCtrl, whereas the cytostatic effect of Adp14 was similar to that of AdCtrl in MS-1 cells (IC₅₀ of AdCtrl/IC₅₀ of Adp14 = 0.79 ± 0.41; Fig. 3 ).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Adp16- or Adp14-related cytotoxicity in mesothelioma cells. H28 and MS-1 mesothelioma cells were infected with varying doses of Adp16, Adp14, or AdCtrl, incubated for 7 days, and cytotoxicity determined using a colorimetric assay as described in "Materials and Methods." The IC50s for Adp14, Adp16, and AdCtrl were calculated for each cell line assuming the survival rate of untreated cells was 100%. Results shown are the ratios of the IC50 after Adp14 or Adp16 infection relative to the IC50s of AdCtrl in each cell line (fold difference in toxicity = IC50 of AdCtrl/IC50 of Adp14 or Adp16). Results were derived from the mean of three determinations ± SD.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Influence of p14ARF gene replacement on CPE of ONYX-015 in H28 mesothelioma cells. ONYX-015 significantly reduced the viable cell numbers in both samples concurrently treated with either Adp14 or AdCtrl (P < 0.001 in both groups). Comparing with AdCtrl, Adp14 alone significantly inhibited the growth of the H28 cells (P = 0.001). Paradoxically, the viable cell number in the sample treated with both Adp14 and ONYX-015 was significantly higher than that treated with both AdCtrl and ONYX-015 (P = 0.01). Results are expressed as the mean of three determinations ± SD.

	Discussion

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The mechanisms involved in viral replication are somewhat similar to those in tumor cell proliferation. A major function of the early proteins of various DNA viruses is to provoke the infected cell to enter the cell cycle and progress to S phase. In this phase of the cell cycle, a virus can take advantage of the host’s DNA replication machinery to duplicate its own DNA. The E1a protein is necessary for adenovirus to drive cells toward S phase. E1a binds pRB and related proteins and, thus, liberates E2F. E2F is essential for S phase entry and is also able to activate transcription from the E2 region of the viral genome, a region that encodes a DNA polymerase and other proteins essential for viral replication (20) . However, E2F also leads to increased transcription of p14^ARF. p14^ARF inhibits MDM2 activity and, thus, allows p53 to accumulate. This in turn can cause growth arrest, through induction of the cell cycle inhibitor p21^WAF (15 , 19) , or it can provoke apoptosis through induction of Bax and by other means. Growth arrest and apoptosis early in infection would certainly reduce viral yield. On the other hand, adenovirus produces E1b 55K, the viral version of MDM2, to inhibit p53 function and, thus, allows the virus to replicate efficiently. E1b 55K can bind p53 and prevent transcriptional activation and exports p53 to the cytoplasm for degradation (20) . ONYX-015, an E1b gene-attenuated adenovirus, should therefore replicate only in p53-deficient cells but not in normal cells. Six cancer cell lines (HCT116, A549, G401, HLaC, McF-7, U87) in which the p53 gene is wild type, however, have been shown to support replication of ONYX-015 (21) . Some of these cells express human papillomavirus E6, a protein known to target p53 for destruction (20) . In the other cases, where ONYX-015 replicates efficiently even in cancer cells, retaining wild-type p53 gene may be attributable to the loss of p14^ARF and subsequent functional inactivation of p53.

Disruption of the p53 pathway in tumor cells, such as H28, H513, and 211H, because of the lack of p14^ARF expression promotes the inhibition of p53 by MDM2, thus allowing ONYX-015 replication regardless of the presence of wild-type p53 gene. The relatively higher titer of ONYX-015 required (2) for cytolysis suggests that p53 functions in these cells are not completely abrogated. In our previous work (15) , we showed that the replacement of p14^ARF gene in these mesothelioma cell lines restores p53 function and results in cell cycle arrest and/or apoptotic cell death. In the present study, MS-1 cells, which express the p14^ARF protein, were resistant to both ONYX-015 and Adp14. Craig et al. (8) reported previously that effects of adenovirus-mediated p16^INK4A expression on cell cycle arrest were determined by endogenous p16^INK4A and pRB status in human cancer cells. p16^INK4A-mediated cytostatic effect is tightly associated with the presence of functional pRB in human cancer cells. Although we did not examine the genetic status of pRB in our cells tested, the assay for p16^INK4A-mediated cytostatic effect in MS-1 and H28 suggested that both cell lines have functional pRB. The difference in sensitivity to Adp14 between these two cells was not related to the status of RB gene. If the loss of p14^ARF leads to the sensitivity of H28 cells to ONYX-015, the replacement of p14^ARF gene should inhibit the CPE of ONYX-015 in this cell. To minimize the cytostatic effect resulting from overexpression of p14^ARF, a lower titer (MOI of 10) of Adp14 was used for gene replacement. At this titer, we found that Adp14, although still having minimal cytostatic effect in H28 cells, was able to attenuate the CPE of ONYX-015 in the cell tested. These results, to a great extent, indicate that the p14^ARF protein can modulate the CPE of ONYX-015 in a cell line with wild-type p53. Reintroduction of p14^ARF into H28 cells did not appear to prevent wild-type virus replication, indicating that the cells are still dividing.

MS-1 appears to have higher levels of p14^ARF and p53 protein (Fig. 2A) , suggesting that there might be some defect in the p53 pathway in MS-1 cells. To explore the response of p53 pathway in MS-1 cells to ONYX-015 infection, we monitored the protein levels of p14^ARF, p21^WAF1, and p53 in a time course study. The p14^ARF, p21^WAF1, and p53 protein levels were found significantly increased at 12 and 24 h after the adenoviral infection, indicating that the p53 pathway in MS-1 cells responded normally to oncogene E1A (Fig. 2B) . Furthermore, we checked MDM2 protein level in MS-1 cells by immunoblotting. MS-1 was found to contain higher levels of MDM2 than H28 cells (data not shown). We hypothesize that the higher MDM2 levels in MS-1 cells lead to the higher levels of p14^ARF, and its inhibition of MDM2 ubiquitin ligase activity is sufficient to stabilize p53 protein (22) . Thus, the levels of p14^ARF and p53 protein are higher in MS-1 cells. Therefore, although the protein levels of p14^ARF and p53 in MS-1 cells are higher than in normal cells, the p53 pathway in MS-1 cells responds normally to oncogenes such as E1A. MS-1 is a unique mesothelioma cell line which has only p16 (Exon 1) deletion but still has intact and functional p14^ARF. This phenomenon was confirmed by PCR, reverse transcription-PCR, and immunoblotting.

We have shown that ONYX-015 infection can lead to a normal response of p53 pathways in MS-1 cells. Previously, we and others have shown that normal cells are resistant to ONYX-015, whereas tumor cells with nonfunctional p53 are sensitive to this adenovirus (3 , 21) . Recently, it was shown that this protective effect against ONYX-015 by re-expression of p14^ARF is p53 dependent, indicating that a functional p14^ARF-p53 pathway is required for inhibiting ONYX-015 replication (23) .

Mutations in p53 genes occur in 60% of all human cancers. Loss of p14^ARF may well account for a significant proportion of the remaining tumors. A recent survey of human tumor-derived cell lines revealed that all cell lines examined that retained wild-type p53 had lost expression of p14^ARF (24) . Our results suggest that these tumors, including MPM, may be potential candidates for ONYX-015 therapy. Because ONYX-015 has been reported to work synergistically with chemotherapy in tumor cells (3 , 21) , additional studies to test the effect of ONYX-015 in combination with conventional chemotherapeutic agent(s) in human mesothelioma cells are currently underway in our laboratory.

	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 To whom requests for reprints should be addressed, at Section of General Thoracic Surgery, University of California, San Francisco, Campus Box 1674, San Francisco, CA 94115. Phone: (415) 885-3882; Fax: (415) 353-9525; E-mail: jablonsd{at}surgery.ucsf.edu

² The abbreviations used are: MPM, malignant pleural mesothelioma; pRB, retinoblastoma protein; ARF, alternate reading frame; CPE, cytopathic effect; WTD, wild-type D; MOI, multiplicity of infection; CMV, cytomegalovirus; GFP, green fluorescent protein.

Received 7/31/00. Accepted 6/21/01.

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