P14arf Modulates the Cytolytic Effect of ONYX-015 in Mesothelioma Cells with Wild-Type P53

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[CANCER RESEARCH 61, 5959–5963, August 15, 2001] Advances in Brief p14ARF Modulates the Cytolytic Effect of ONYX-015 in Mesothelioma Cells with Wild-type p53

Cheng-Ta Yang, Liang You, Kazutsugu Uematsu, Che-Chung Yeh, Frank McCormick, and David M. Jablons1 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 lung cancer cells lacking functional p53 with comparable efficiency to wild-type adenovirus (2, 3). In mesothelioma, unlike other adult ONYX-015 has been reported to kill selectively tumor cells lacking malignancies, genetic alterations in p53 are rare (4). Homozygous functional p53. Genetic alterations of INK4a/ARF locus, which is a pre- deletions of INK4a/ARF locus, however, have been shown to be the dominant event in malignant pleural mesothelioma, may result in loss of Ͼ p14ARF and subsequent disruption of p53 pathway in cancer cells. In the predominant events which occur at a frequency of 70% in this present study, ONYX-015 was able to kill three mesothelioma cell lines malignancy (5, 6). The INK4a/ARF locus (7) on human chromosome (H28, H513, and 211H) with wild-type p53 but lacking p14ARF.Incon- 9p21 plays an important role in both the pRB and p53 tumor sup- trast, MS-1 mesothelioma cells, which expressed both p53 and p14ARF, pressor pathways by encoding two distinct proteins translated from were resistant to ONYX-015. Introducing p14ARF gene into the H28 cell, a alternatively spliced mRNAs. p16INK4a has been biochemically char- mesothelioma cell without p14ARF expression, significantly increased the acterized as a protein that specifically binds to and inhibits cyclin- resistance of this cell line to the cytolytic effect of ONYX-015. Our results dependent kinase-4/6. Thus, p16INK4a regulates pRB phosphorylation suggest that human mesotheliomas with wild-type p53 yet lacking p14ARF ARF and induces cell cycle arrest in G1 phase (8). p14 plays a role as are potential candidates for ONYX-015 therapy. a negative regulator of MDM2, interfering with MDM2-mediated shuttling and degradation of p53 (9–11). A single mutational event at Introduction the INK4a/ARF locus therefore has the potential to disrupt both pRB ARF MPM2 is an asbestos-related malignancy characterized by rapidly and p53 tumor suppressor pathways. In addition, the p14 gene progressive and diffusely local growth, late metastases, and poor promoter is a CpG island that can be silenced by DNA methylation prognosis. Approximately 3,000 patients are diagnosed with MPM in (12). These genetic alterations may result in a loss of negative regu- the United States annually. The incidence of this disease worldwide is lation of MDM2. Because MDM2 has functional similarity to the ARF rising and is expected to peak in the next 2 decades (1). Chemotherapy adenoviral E1B 55K protein, lack of p14 may thus prevent a or radiation therapy, alone or in combination, is not effective, and the normal p53 response to viral infection, thereby allowing ONYX-015 majority of patients with MPM die within 2 years regardless of replication. In the present study, we showed that ONYX-015 killed treatment (1). Considering the uniformly fatal outcome and lack of effectively mesothelioma cells with normal p53 gene but lacking ARF ARF effective treatment, the development of novel treatment strategies for p14 expression. Introducing the p14 gene into human mesothe- ARF MPM given the lack of response with conventional therapies is lioma cells lacking p14 expression by use of an adenoviral vector therefore needed. Replicating oncolytic virus is a promising new significantly increased the resistance of cells to the CPE of ONYX- modality for cancer treatment. The strategy of this therapy is to 015. This study suggests that ONYX-015 may be an effective treat- ARF develop viruses capable of selectively infecting malignant tumors ment for tumors retaining wild-type p53 but lacking p14 , such which can spread and destroy malignant tumors without deleterious as MPM. effects in normal tissues. The key to the development of such viruses Materials and Methods is the identification of viral genes whose deletion or modification enables tumor-specific cell killing. ONYX-015, a conditional replica- Cell Lines and Cell Culture. H28 (ATCC CRL-5820), H513 (ATCC tion-competent adenovirus lacking E1b 55 kDa gene, is a promising CRL-5830), and MSTO-211H (ATCC CRL-2081, 211H) mesothelioma cells agent based on this strategy. ONYX-015 contains an 827-bp deletion and Met5A (ATCC CRL-9444) transformed mesothelium cells were obtained in the E1b region and a point mutation at codon 2022 that generates from American Type Culture Collection. MS-1 mesothelioma cell line has a stop codon. This genetic design takes advantage of the fact that been described previously (13). All were cultured in RPMI 1640 complete adenovirus E1b 55k binds and thus inactivates wild-type p53 protein. media containing 10% FCS. This binding/inactivation is essential to virus replication. ONYX-015 CPE Assay. ONYX-015 and WTD were supplied by ONYX Pharmaceu- ticals (Richmond, CA). WTD is identical to ONYX-015 except in the E1B, cannot replicate in normal cells but can replicate in tumor cells 55-kDa gene region where the original, wild-type sequence is present. Cells lacking functional p53 (2). ONYX-015 has been shown to kill cervi- (1 ϫ 105 cells/well) were plated in replicate 6-well plates, incubated overnight cal, colon, pancreatic carcinoma cells, glioma cells, and non-small cell 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% Received 7/31/00; accepted 6/21/01. FCSfor4hat37°C. Then cells were incubated in complete medium containing The costs of publication of this article were defrayed in part by the payment of page 10% FCS, and plates were monitored daily for CPE. The assay was terminated charges. This article must therefore be hereby marked advertisement in accordance with when essentially total cytolysis was observed in cultures infected with WTD at 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 an MOI of 0.1. The plates were then stained with crystal violet (0.5% in 20% Surgery, University of California, San Francisco, Campus Box 1674, San Francisco, CA methano; Sigma Chemical Co., St. Louis, MO) for analysis. 94115. Phone: (415) 885-3882; Fax: (415) 353-9525; E-mail: [email protected]. Adenoviral Vectors. The adenoviral vectors for gene expression studies 2 The abbreviations used are: MPM, malignant pleural mesothelioma; pRB, retino- were constructed by using the AdEasy System (14). The p14ARF and p16INK4A blastoma protein; ARF, alternate reading frame; CPE, cytopathic effect; WTD, wild-type D; MOI, multiplicity of infection; CMV, cytomegalovirus; GFP, green fluorescent cDNA were kindly provided by Dr. K. Vousden (National Cancer Institute, protein. Frederick, MD) and Dr. D. Beach (Institute of Child Health, London, England, 5959

United Kingdom). Briefly, the gene of interest was first cloned into a shuttle of 1 within 10 days. Only MS-1 was resistant to ONYX-015 up to vector, pAdTrack-CMV, which contained CMV promoters and a GFP gene. MOI of 1 (Fig. 1). The resultant plasmid was subsequently cotransformed into Escherichia coli p53, p21WAF1, and p14ARF Expression in Mesothelioma Cells. ARF BJ5183 cells with an adenoviral backbone plasmid, pAdEasy-1. p14 and Previously, we have reported the absence of p14ARF and p16INK4A INK4A p16 recombinant adenoviruses (Adp14 and Adp16, respectively) were expression in H28, H513, and 211H cells (15). In the present study, then generated in 293 cells (E1-transformed human embryonic kidney cells) expression of endogenous p14ARF was detected by immunoblotting in and identified by PCR analysis of the DNA samples prepared from the cell culture supernatant. The recombinant adenovirus, AdCtrl, which carries a GFP MS-1 cells (Fig. 2A). We have detected homozygous deletion of ␣ ␤ gene regulated by the CMV promoter, was constructed with pAdTrack and exons 1 and 1 of INK4a/ARF locus in both H28 and 211H cells. pAdEasy-1 and used as a control in these experiments. Loss of exon 1␣ only (but not 1␤) was found in MS-1 cells (data not ARF Immunoblotting. Cells (1 ϫ 106) were plated in 10-cm dishes and incu- shown). The transcription of p14 (exon 1␤) in MS-1 cells and the bated for4hat37°C. Cells were then mock infected or infected with either absence of p14 ARF transcripts in H28, H513, and 211H cells was AdCtrl or Adp14 (MOI of 50 each) and incubated for 72 h. Cell samples were confirmed by reverse transcription-PCR (data not shown). Moreover, washed twice with PBS, scraped off the plates, and lysed in cell lysis buffer [50 the absence of p16 as well as p14ARF expression in H28 cells was also mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, revealed by Waheed et al. (16). To investigate adenovirus-mediated and 0.1% SDS]. Whole cell lysates were boiled, and the protein concentration p14ARF expression in mesothelioma cells, the H28 cell line was was determined using the Bradford assay (Bio-Rad, Hercules, CA). Balanced infected with Adp14 or AdCtrl. Forty-eight h after transfection at MOI amounts of cell proteins (20–40 ␮g) were boiled again after the addition of mercaptoethanol and bromphenol blue and fractionated by SDS-PAGE in of 50, fluorescent microscopic examination revealed that nearly 100% 4–20% linear gradient acrylamide gel (Ready-Gel; Bio-Rad). After transfer- of the cells became GFP positive. As illustrated in Fig. 2A, endoge- ARF ring the proteins onto Immobilon-P (Millipore, Bedford, MA), the membranes nous p14 expression was absent in H28 mesothelioma cells. After ARF were blocked in 5% nonfat milk powder, 0.2% Tween 20 in TBS overnight at Adp14 infection, strong expression of p14 was detected by im- 4°C, and incubated with the primary antibody for1hatroom temperature. munoblotting. Because p14ARF functions to stabilize p53 by inhibiting Membranes were then washed in Tween 20 in TBS for 5 min three times. MDM2, we looked next for the effect of p14ARF overexpression on Primary antibodies for p14ARF (C-18), p21WAF1 (C-19), and p53 (DO-1) were p53 expression. The expression of endogenous p53 in H28 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Depending on the markedly lower than that in MS-1. Transfection with Adp14 at MOI experiment, goat antimouse horseradish peroxidase and donkey antigoat horse- of 50 increased markedly the amount of p14ARF expression and also radish peroxidase were used as secondary antibodies (Santa Cruz Biotechnol- increased the expression of p53 in H28 (Fig. 2A). Treatment with ogy). Antibody binding was visualized using Chemiluminescence Luminol ARF reagents (Santa Cruz Biotechnology). AdCtrl did not have significant effect on p14 or p53 expression in Cytotoxicity Assays. Cells were plated in triplicate on 96-well culture this cell line (15). ARF WAF1 plates (500 cells/well) and incubated overnight at 37°C. Cells were then In a time course study, p14 , p21 , and p53 expression in exposed to varying concentrations of AdCtrl, Adp14, or Adp16 and incubated MS-1 cells at 0, 3, 6, 9, 12, and 24 h after ONYX-015 infection (MOI for 7 days at 37°C. A colorimetric assay was performed as described previ- of 1) was determined by Western blot analysis (Fig. 2B). A significant ously (15). Briefly, cells were fixed in 10% trichloroacetic acid for 1 h, washed increase of expression of p14ARF, p21WAF1, and p53 was noticed at 12 five times with water, and allowed to air dry. Cells were then stained for 30 and 24 h after adenoviral infection. A decline of protein levels of min with 0.4% sulforhodamine B (Sigma Chemical Co.), dissolved in 1% p14ARF, p21WAF1, and p53 was observed at 24 h after the infection. acetic acid, and rinsed five times with 1% acetic acid to remove unbound dye. Cytostatic Effect of Adenovirus-mediated p14ARF or p16INK4A Bounded dye was then solubilized with 10 mM unbuffered Tris-base (pH 10.5) Expression in H28 and MS-1 Mesothelioma Cells. Both p14ARF for 5 min. Visual absorbance (A595) was obtained using a kinetic microplate INK4A reader (Vmax; Molecular Devices, Sunnyvale, CA), which was used as a and p16 function as the tumor suppressors, and they have been targeted for gene-replacement strategies for cancer therapy (7, 8, 15, 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 17, 18). We additionally studied the cytostatic effect of gene replace- ARF INK4A relative differences in IC50s were calculated by dividing the IC50 of cells ment with p14 or p16 in H28, the cell lacking expression of infected with Adp14 or Adp16 by the IC50 of cells infected with AdCtrl for each cell line. Measurement of Cell Viability. H28 cells (1 ϫ 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%FCSfor4hat37°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 Ϯ SD. Statistical comparisons were made by two-sided t test. A value of P Ͻ 0.05 was accepted as significant.

Results 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 conforma- Fig. 1. CPE of ONYX-015 on mesothelioma cells. ONYX-015 (015) lysed H28 cells tion polymorphism, and no mutations in exons 5–8ofthep53 gene 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 were detected (data not shown). ONYX-015, however, can effectively used are shown to the right, the infectious agents are shown to the left, and the titers are lyse H28 at MOI of 0.1, and both H513 and 211H were lysed at MOI above. 5960

with either Adp14 or AdCtrl (16.33 Ϯ 2.75 versus 34.0 Ϯ 1.5 or 8.83 Ϯ 0.76 versus 41.5 Ϯ 0.5 ϫ 104 cell/well, respectively; P Ͻ 0.001 in both comparisons). The viable cell number in the sample treated with Adp14 and ONYX-015 was significantly higher than that treated with AdCtrl and ONYX-015 (16.33 Ϯ 2.75 versus 8.83 Ϯ 0.76 ϫ 104 cell/well, respectively; P ϭ 0.01, on triplicate samples).

Discussion Functional inactivation of pRB and p53 tumor suppressor pathways appears to be a fundamental requirement for the development of most

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 ARF WAF1 Fig. 3. Adp16- or Adp14-related cytotoxicity in mesothelioma cells. H28 and MS-1 of p14 , p21 , and p53 in MS-1 cells at 3, 6, 9, 12, and 24 h after ONYX-015 mesothelioma cells were infected with varying doses of Adp16, Adp14, or AdCtrl, infection. The time periods after the infection are shown above their corresponding lanes, incubated for 7 days, and cytotoxicity determined using a colorimetric assay as described and the antibodies are shown to the right. 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 ARF INK4A ϭ both the p14 and p16 proteins. We also studied MS-1, a cell each cell line (fold difference in toxicity IC50 of AdCtrl/IC50 of Adp14 or Adp16). line with p14ARF expression but deficient in p16INK4A, by examining Results were derived from the mean of three determinations Ϯ SD. 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 ϭ Ϯ AdCtrl/IC50 of Adp14 0.79 0.41; Fig. 3). Treatment with p14ARF Recombinant Adenoviruses Induced Resistance to the CPE of ONYX-015 in H28 Mesothelioma Cells. To examine if the restoration of the p14ARF function in H28 cells would increase the resistance of the cell to ONYX-015-induced cytolytic effect, we infected H28 with combinations of either WTD or ONYX-015 (MOI of 1 each) and Adp14 or AdCtrl (MOI of 10 each). Although infection with Adp14 alone significantly inhibited the growth of the H28 cells compared with AdCtrl (41.5 Ϯ 0.5 versus 34.0 Ϯ 1.5 ϫ 104 cell/well, respectively; P ϭ 0.001, in triplicate samples), the Adp14 attenuated the cytolytic effect of ONYX-015 (Fig. 4). When H28 cells were coinfected with WTD and AdCtrl, a complete cytolysis was observed at day 9, and there were ϳ5000 ARF viable cells in the well coinfected with WTD and Adp14 (in triplicate) Fig. 4. Influence of p14 gene replacement on CPE of ONYX-015 in H28 mesothelioma cells. ONYX-015 significantly reduced the viable cell numbers in both samples at the same time point. An additional 36 h of incubation was required concurrently treated with either Adp14 or AdCtrl (P Ͻ 0.001 in both groups). Comparing (time course not shown) for the latter to reach the complete cell lysis. with AdCtrl, Adp14 alone significantly inhibited the growth of the H28 cells (P ϭ 0.001). Paradoxically, the viable cell number in the sample treated with both Adp14 and ONYX- As shown in Fig. 4, ONYX-015 significantly reduced the viable cell 015 was significantly higher than that treated with both AdCtrl and ONYX-015 numbers compared with mock infection in both samples coinfected (P ϭ 0.01). Results are expressed as the mean of three determinations Ϯ SD. 5961

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. p14ARF MODULATES ONYX-015 EFFECT IN MESOTHELIOMAS human cancers. In normal cells, pRB regulates cell proliferation by of ONYX-015 in this cell. To minimize the cytostatic effect resulting binding and sequestering E2F transcriptional factors essential for from overexpression of p14ARF, a lower titer (MOI of 10) of Adp14 progression to S phase. These transcriptional factors are released in was used for gene replacement. At this titer, we found that Adp14, late G1 phase by the cyclin D-dependent kinase-mediated phospho- although still having minimal cytostatic effect in H28 cells, was able rylation of pRB, thereby allowing cells to enter S phase (7, 19). to attenuate the CPE of ONYX-015 in the cell tested. These results, to Defects in the pRB pathways, such as mutations in the RB gene or loss a great extent, indicate that the p14ARF protein can modulate the CPE of p16INK4A, lead to the deregulation of the E2F transcription factors. of ONYX-015 in a cell line with wild-type p53. Reintroduction of Hyperactivation of E2F results in increased transcription of p14ARF, p14ARF into H28 cells did not appear to prevent wild-type virus and p14ARF stabilizes p53 by promoting MDM2 degradation. Accu- replication, indicating that the cells are still dividing. mulation of p53 can cause growth arrest and induce apoptosis and, MS-1 appears to have higher levels of p14ARF and p53 protein (Fig. thus, prevent uncontrolled cell proliferation. Therefore, most tumors 2A), suggesting that there might be some defect in the p53 pathway in having defects in pRB pathways should harbor defects in p53 path- MS-1 cells. To explore the response of p53 pathway in MS-1 cells to ways as well. ONYX-015 infection, we monitored the protein levels of p14ARF, The mechanisms involved in viral replication are somewhat similar p21WAF1, and p53 in a time course study. The p14ARF, p21WAF1, and to those in tumor cell proliferation. A major function of the early p53 protein levels were found significantly increased at 12 and 24 h proteins of various DNA viruses is to provoke the infected cell to after the adenoviral infection, indicating that the p53 pathway in MS-1 enter the cell cycle and progress to S phase. In this phase of the cell cells responded normally to oncogene E1A (Fig. 2B). Furthermore, we cycle, a virus can take advantage of the host’s DNA replication checked MDM2 protein level in MS-1 cells by immunoblotting. MS-1 machinery to duplicate its own DNA. The E1a protein is necessary for was found to contain higher levels of MDM2 than H28 cells (data not adenovirus to drive cells toward S phase. E1a binds pRB and related shown). We hypothesize that the higher MDM2 levels in MS-1 cells proteins and, thus, liberates E2F. E2F is essential for S phase entry lead to the higher levels of p14ARF, and its inhibition of MDM2 and is also able to activate transcription from the E2 region of the viral ubiquitin ligase activity is sufficient to stabilize p53 protein (22). genome, a region that encodes a DNA polymerase and other proteins Thus, the levels of p14ARF and p53 protein are higher in MS-1 cells. essential for viral replication (20). However, E2F also leads to in- Therefore, although the protein levels of p14ARF and p53 in MS-1 creased transcription of p14ARF. p14ARF inhibits MDM2 activity and, cells are higher than in normal cells, the p53 pathway in MS-1 cells thus, allows p53 to accumulate. This in turn can cause growth arrest, responds normally to oncogenes such as E1A. MS-1 is a unique through induction of the cell cycle inhibitor p21WAF (15, 19), or it can mesothelioma cell line which has only p16 (Exon 1␣) deletion but still provoke apoptosis through induction of Bax and by other means. has intact and functional p14ARF. This phenomenon was confirmed by Growth arrest and apoptosis early in infection would certainly reduce PCR, reverse transcription-PCR, and immunoblotting. viral yield. On the other hand, adenovirus produces E1b 55K, the viral We have shown that ONYX-015 infection can lead to a normal version of MDM2, to inhibit p53 function and, thus, allows the virus response of p53 pathways in MS-1 cells. Previously, we and others to replicate efficiently. E1b 55K can bind p53 and prevent transcrip- have shown that normal cells are resistant to ONYX-015, whereas tional activation and exports p53 to the cytoplasm for degradation tumor cells with nonfunctional p53 are sensitive to this adenovirus (3, (20). ONYX-015, an E1b gene-attenuated adenovirus, should there- 21). Recently, it was shown that this protective effect against ONYX- fore replicate only in p53-deficient cells but not in normal cells. Six 015 by re-expression of p14ARF is p53 dependent, indicating that a cancer cell lines (HCT116, A549, G401, HLaC, McF-7, U87) in functional p14ARF-p53 pathway is required for inhibiting ONYX-015 which the p53 gene is wild type, however, have been shown to support replication (23). replication of ONYX-015 (21). Some of these cells express human Mutations in p53 genes occur in ϳ60% of all human cancers. Loss papillomavirus E6, a protein known to target p53 for destruction (20). of p14ARF may well account for a significant proportion of the In the other cases, where ONYX-015 replicates efficiently even in remaining tumors. A recent survey of human tumor-derived cell lines cancer cells, retaining wild-type p53 gene may be attributable to the revealed that all cell lines examined that retained wild-type p53 had loss of p14ARF and subsequent functional inactivation of p53. lost expression of p14ARF (24). Our results suggest that these tumors, Disruption of the p53 pathway in tumor cells, such as H28, H513, including MPM, may be potential candidates for ONYX-015 therapy. and 211H, because of the lack of p14ARF expression promotes the Because ONYX-015 has been reported to work synergistically with inhibition of p53 by MDM2, thus allowing ONYX-015 replication chemotherapy in tumor cells (3, 21), additional studies to test the regardless of the presence of wild-type p53 gene. The relatively higher effect of ONYX-015 in combination with conventional chemothera- titer of ONYX-015 required (2) for cytolysis suggests that p53 func- peutic agent(s) in human mesothelioma cells are currently underway tions in these cells are not completely abrogated. In our previous work in our laboratory. (15), we showed that the replacement of p14ARF gene in these mesothelioma cell lines restores p53 function and results in cell cycle arrest References and/or apoptotic cell death. In the present study, MS-1 cells, which 1. Bueno, R. Mesothelioma clinical presentation. Chest, 116: 444S–445S, 1999. ARF express the p14 protein, were resistant to both ONYX-015 and 2. Bischoff, J. R., Kirn, D. H., Williams, A., Heise, C., Horn, S., Muna, M., Ng, L., Nye, Adp14. Craig et al. (8) reported previously that effects of adenovirus- J. A., Sampson-Johannes, A., Fattaey, A., and McCormick, F. An adenovirus mutant INK4A that replicates selectively in p53-deficient human tumor cells. Science (Wash. DC), mediated p16 expression on cell cycle arrest were determined by 274: 373–376, 1996. endogenous p16INK4A and pRB status in human cancer cells. 3. You, L., Yang, C. T., and Jablons, D. M. ONYX-015 works synergistically with p16INK4A-mediated cytostatic effect is tightly associated with the chemotherapy in lung cancer cell lines and primary cultures freshly made from lung cancer patients. Cancer Res., 60: 1009–1013, 2000. presence of functional pRB in human cancer cells. Although we did 4. Mor, O., Yaron, P., Huszar, M., Yellin, A., Jakobovitz, O., Brok-Simoni, F., Rechavi, not examine the genetic status of pRB in our cells tested, the assay for G., and Reichert, N. Absence of p53 mutations in malignant mesotheliomas. Am. J. p16INK4A-mediated cytostatic effect in MS-1 and H28 suggested that Respir. Cell Mol. Biol., 16: 9–13, 1997. 5. Cheng, J. Q., Jhanwar, S. C., Klein, W. M., Bell, D. W., Lee, W. C., Altomare, D. A., both cell lines have functional pRB. The difference in sensitivity to Nobori, T., Olopade, O. I., Buckler, A. J., and Testa, J. R. p16 alterations and deletion Adp14 between these two cells was not related to the status of RB mapping of 9p21–p22 in malignant mesothelioma. Cancer Res., 54: 5547–5551, ARF 1994. gene. If the loss of p14 leads to the sensitivity of H28 cells to 6. Prins, J. B., Williamson, K. A., Kamp, M. M., Van Hezik, E. J., Van der Kwast, T. H., ONYX-015, the replacement of p14ARF gene should inhibit the CPE Hagemeijer, A., and Versnel, M. A. The gene for the cyclin-dependent-kinase-4 5962

inhibitor, CDKN2A, is preferentially deleted in malignant mesothelioma. Int. J. 15. Yang, C. T., You, L., Yeh, C. C., Chang, J. W., Zhang, F., McCormick, F., and Cancer, 75: 649–653, 1998. Jablons, D. M. Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma 7. Chin, L., Pomerantz, J., and DePinho, R. A. The INK4a/ARF tumor suppressor: one cells. J. Natl. Cancer Inst. (Bethesda), 92: 636–641, 2000. gene–two products–two pathways. Trends Biochem. Sci., 23: 291–296, 1998. 16. Waheed, I., Guo, Z. S., Chen, G. A., Weiser, T. S., Nguyen, D. M., and Schrump, 8. Craig, C., Kim, M., Ohri, E., Wersto, R., Katayose, D., Li, Z., Choi, Y. H., Mudahar, D. S. Antisense to SV40 early gene region induces growth arrest and apoptosis in B., Srivastava, S., Seth, P., and Cowan, K. Effects of adenovirus-mediated p16INK4A T-antigen-positive human pleural mesothelioma cells. Cancer Res., 59: 6068–6073, expression on cell cycle arrest are determined by endogenous p16 and Rb status in 1999. human cancer cells. Oncogene, 16: 265–272, 1998. 17. Frizelle, S. P., Grim, J., Zhou, J., Gupta, P., Curiel, D. T., Geradts, J., and Kratzke, 9. Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, R. A. Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, L., Potes, J., Chen, K., Orlow, I., Lee, H. W., Cordon-Cardo, C., and DePinho, R. A. cell death, tumor suppression and tumor regression. Oncogene, 16: 3087–3095, 1998. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neu- 18. Jin, X., Nguyen, D., Zhang, W. W., Kyritsis, A. P., and Roth, J. A. Cell cycle arrest tralizes MDM2’s inhibition of p53. Cell, 92: 713–723, 1998. and inhibition of tumor cell proliferation by the p16INK4 gene mediated by an adenovirus vector. Cancer Res., 55: 3250–3253, 1995. 10. Zhang, Y., Xiong, Y., and Yarbrough, W. G. ARF promotes MDM2 degradation and 19. Sherr, C. J. Cancer cell cycles. Science, 274: 1672–1677, 1996. stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor 20. McCormick, F. Cancer therapy based on p53. Sci. Am., 5: 139–144, 1999. suppression pathways. Cell, 92: 725–734, 1998. 21. Heise, C., Sampson-Johannes, A., Williams, A., McCormick, F., Von Hoff, D. D., and 11. Piette, J., Neel, H., and Marechal, V. Mdm2: keeping p53 under control. Oncogene, Kirn, D. H. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific 15: 1001–1010, 1997. cytolysis and antitumoral efficacy that can be augmented by standard chemothera- 12. Robertson, K. D., and Jones, P. A. The human ARF cell cycle regulatory gene peutic agents. Nat. Med., 3: 639–645, 1997. promoter is a CpG island which can be silenced by DNA methylation and down- 22. Woods, D. B., and Vousden, K. H. Regulation of p53 function. Exp. Cell Res., 264: regulated by wild-type p53. Mol. Cell. Biol., 18: 6457–6473, 1998. 56–66, 2001. 13. Hsu, S. M., Hsu, P. L., Zhao, X., Kao-Shan, C. S., and Whang-Peng, J. Establishment 23. Ries, S. J., Brandts, C. H., Chung, A. S., Biederer, C. H., Hann, B. C., Lipner, E. M., of human mesothelioma cell lines (MS-1, -2) and production of a monoclonal McCormick, F., and Korn, W. M. Loss of p14ARF in tumor cells facilitates replica- antibody (anti-MS) with diagnostic and therapeutic potential. Cancer Res., 48: 5228– tion of the adenovirus mutant dl1520 (ONYX-015). Nat. Med., 6: 1128–1133, 2000. 5236, 1988. 24. Stott, F. J., Bates, S., James, M. C., McConnell, B. B., Starborg, M., Brookes, S., 14. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. A Palmero, I., Ryan, K., Hara, E., Vousden, K. H., and Peters, G. The alternative simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. product from the human CDKN2A locus, p14(ARF), participates in a regulatory USA, 95: 2509–2514, 1998. feedback loop with p53 and MDM2. EMBO J., 17: 5001–5014, 1998.

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Cheng-Ta Yang, Liang You, Kazutsugu Uematsu, et al.

Cancer Res 2001;61:5959-5963.

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