Replication of an E1B 55-Kilodalton Protein-Deficient Adenovirus (ONYX-015) Is Restored by Gain-of-Function Rather than Loss-of-Function p53 Mutants

ONYX-015 (dl1520) is an E1B 55-kilodalton protein-deficientreplicating adenovirus that is currently in clinical trialsas an antitumor agent. On the basis of the observation thatthe E1B 55kD gene product is able to bind to and inactivatep53, ONYX-015's mechanism of action is proposed to involve selectivereplication in and killing of p53-deficient cells. While itsefficacy as a therapeutic agent appears evident, the virus'smechanism of cellular selectivity, including a possible roleof p53 in this regard, is less clear. Indeed, there have beena number of recent reports suggesting that the p53 status oftarget cells does not reliably predict ONYX-015 replicationor cell killing. To address the role of p53 in ONYX-015 selectivity,we have undertaken a rigorous analysis of the behavior of thisvirus in small airway-derived primary human epithelial cellsexpressing either dominant-negative or gain-of-function mutantp53 genes. Examination of small airway epithelial cells expressinga variety of p53 mutant alleles revealed that while all wereable to inhibit endogenous p53 activity, only one allele examined,248W, demonstrated a markedly increased ability to facilitateONYX-015 replication. This allele is a member of a group ofp53 mutants (know as class I mutants) characterized by retentionof global structural conformation but loss of DNA-binding activity.These observations indicate that the nature of the p53 mutationaffects ONYX-015 replication, help reconcile disparate publishedfindings, and may provide criteria by which to direct clinicalapplication of ONYX-015.

INTRODUCTION

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Introduction

The p53 gene is mutated or lacking in at least 50% of humancancers, including multiple tumor types (24, 37). p53 inactivationis typically caused by somatic point mutations, most frequentlyin the region of the gene corresponding to the central, DNA-bindingdomain of the protein. Several of these hotspot mutations resultin disruption of the sequence-specific DNA-binding ability ofp53 (4, 18). On the basis of crystal structure and conformation-specificantibody information, p53 mutants have been divided into twoclasses: class I mutants are those that contain aberrant residuesat the sites of direct DNA-protein contact (thereby inhibitingefficient DNA binding) but otherwise retain structural integrity,whereas class II mutants are those in which the global proteinstructure (tertiary or quaternary) has been disrupted in a mannerthat prevents DNA binding (4, 39). In addition, a third categoryof mutant p53 has been described on the basis of its abilityto inhibit endogenous p53 and corresponds to the C terminusof p53, including the tetramerization domain (38, 44). All threep53 mutant types are known to act as dominant-negative inhibitorsof p53, presumably by interacting with wild-type p53 as partnersin a nonfunctional tetrameric complex.

Some dominant-negative alleles of p53 demonstrate gain-of-functionproperties. For example, expression of certain alleles of mutantp53 in p53 null cells yields increased tumors in animal models(49) and promotes enhanced transformation of established celllines (7, 9) compared with expression of the p53 null controlsalone. In another report, some mutant p53 alleles, but not others,were shown to inhibit a G₂-M spindle checkpoint, resulting inendoreduplication (19). Further evidence of gain of functionwas seen in a murine model in which in vivo expression of amutant p53 (172H, equivalent to human 175H) yielded an alteredtumor spectrum and a dramatically higher rate of tumor metastasisthan that of the null allele (30). Finally, although the mechanismis unknown, recent evidence suggests that the ability of certainp53 mutants to interact with and inhibit the p53-related proteinp73 may contribute to a gain-of-function phenotype (6, 11, 31).

p53 is activated in response to a variety of cellular stresssignals, including DNA damage, aberrant proliferation, nucleotidedeprivation, and hypoxia (40, 43), of which one or more arepredicted to occur following viral infection. Activation ofp53 results in cell growth arrest or apoptosis, depending onthe stimulus and cell type (2, 29, 48). Interestingly, the p53response in epithelial cells (the physiological target of adenovirus)appears to be qualitatively different from that in fibroblastsor transformed cells, with which it has been more extensivelyanalyzed. Specifically, a variety of primary epithelial cells,including prostate, breast, bronchial, and keratinocyte cells,show an attenuated or absent G₁ checkpoint response followingIR exposure (5, 10, 13, 32, 36), the basis of which is not clear.Of note, the steady-state levels of p53 are often higher inepithelial cells, due seemingly to a longer protein half-life(5). The effect of these differences on the epithelial cell'sresponse to adenoviral infection is not known.

Given the prevalence of p53 mutations in human cancers, p53deficiency represents an attractive criterion upon which tobase a tumor-specific cancer therapeutic. To this end, McCormickand colleagues have pioneered the clinical application of agenetically modified adenovirus, dl1520 (termed ONYX-015), thatis proposed to selectively replicate in, and thereby kill, p53-deficienttumor cells (3, 22). Initial clinical studies show promise,particularly when combined with conventional chemotherapeuticcompounds, and the agent is currently being tested in phaseIII clinical trials (26, 27, 34). Originally constructed anddescribed by Barker and Berk (1), ONYX-015 lacks the virallyencoded 55-kDa E1B gene (E1B 55kD). In addition to its knownlate-phase functions, the E1B 55kD protein is known to bindcellular p53, repress its transcriptional activity, and promoteits degradation, thereby leading to an inactivation of p53-mediatedcheckpoints. These latter functions of E1B 55kD are proposedto provide ONYX-015's mechanism of selectivity. Cells containingan intact p53 pathway are thus predicted to inhibit replicationof an E1B 55kD-deficient virus. In contrast, p53-deficient cells,such as those of a tumor, would be expected to allow efficientviral replication and subsequent cell killing.

The proposed basis for ONYX-015's oncolytic specificity hasbeen challenged. While a number of studies support a role forp53 and p53 pathway components in determining viral selectivity(3, 12, 22, 28, 41, 50), others have failed to do so. Indeed,a number of groups have argued that ONYX-015 replication isindependent of the host cell's p53 status (17, 20, 21, 42, 46,47). The reasons for the apparent disparity are not entirelyclear but are likely to arise in part from the different experimentalmodels used. Typically, the adenoviral responses in cells ofsimilar p53 status (but of various cellular backgrounds) havebeen compared with those in cells of differing p53 status. Uncontrolleddifferences in tissue of origin, genetic background, and infectibilityall confound such comparisons. Furthermore, various readoutshave been used as assay endpoints, including cell viability(17, 20), cytopathic effect (CPE) (42), viral burst measuredin PFU/cell (17, 21, 46) and PFU/ml (42), hexon positivity (46),and CPE index (20), thereby further complicating interpretationand cross-comparison. A number of studies have presented modelsin which internally controlled matched pairs of cells have beencompared. Typically, a p53 wild-type tumor cell line has beenstably transfected with a vector expressing dominant-negativep53 or vector-only control to generate a matched pair that canbe assayed for response following viral infection with eitherwild-type or E1B 55kD-deficient adenoviruses (12, 21, 42, 46).While this model represents an improved approach, it, too, suffersfrom experimental limitations. First, numerous studies now indicatethat few, if any, tumor cell lines are truly wild type for p53function; in most cell lines bearing the sequence of wild-typep53, p14ARF is either lacking or silenced (45). This is significantin light of a recent report suggesting that p14ARF is requiredfor selective inhibition of ONYX-015 replication (41). Second,stably transfected cell lines are derived from clonal isolatesthat may have acquired other genetic changes in the selectionprocess. Finally, in all cases only one p53 mutant allele wasexamined, resulting in limited analysis of potential allele-specificdifferences. Furthermore, certain mutant alleles are only weaklydominantly negative, thereby necessitating a clear demonstrationof their loss of endogenous p53 function. Matched cell pairshave also been described in reports of studies using a temperature-sensitiveallele of p53, and phenotypic comparisons were analyzed at restrictiveand permissive temperatures (17, 21). This approach is furtherconfounded by the fact that the E1B 55kD-deficient viruses arethemselves cold sensitive (17, 21, 23).

In this paper, we describe the results of experiments designedto test the role of p53 in modulating E1B 55kD-deficient adenoviralreplication by comparing wild-type control and p53 function-deficientprimary airway epithelial cells, a physiologic target of adenovirus.We used early-passage primary cells to ensure that both efferentand afferent p53 pathways were intact. A variety of p53 mutantalleles were then utilized to render the cells p53 deficient.Nonclonal cell populations were examined, and loss of p53 functionwas demonstrated in cells expressing the various mutant alleles.Our results show that loss of p53 function per se is not sufficientto enable efficient replication of ONYX-015; however, one particularallele did demonstrate a gain of function in rescuing viralreplication.

MATERIALS AND METHODS

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Materials and Methods

Materials. Small airway epithelial cells (SAEC) and cell culture growthmedia were obtained from Clonetics (BioWhittaker, Inc., Walkersville,Md.). Viruses (ONYX-015 and WT-D) were obtained from Onyx Pharmaceuticals,Richmond, Calif. Mutant p53 143A (pBabe-Puro) was obtained fromT. Tlsty (University of California San Francisco). The geneticsuppressor element (GSE) clone was obtained from V. Ossovskaya(University of California San Francisco). The ${Phi}$ NX-ampho retroviralpackaging cell line was obtained from G. Nolan, Stanford, Calif.Anti-p53 antibodies (DO-1, CM-1) were kindly provided by D.Lane, Dundee, Scotland.

Methods. Plasmid construction. p53 mutants 175H and 248W were generated by site-specific mutagenesis,and full-length human cDNAs or the GSE fragment, correspondingto a 94-amino-acid peptide derived from the C terminus of ratp53 (38), were subcloned into either LXSN (Clontech Laboratories,Inc.) or pBabePuro (33) vectors.

Cell culture. Passage 2 SAEC were derived from multiple donors and grown (usingpredefined medium) in cultures according to the manufacturer'srecommendations to approximately 40% confluence prior to infectionwith amphotropic retrovirus with either a vector control ora p53 mutant, as described below. Cells containing the transducedgene were enriched by 48 h of drug selection with either neomycin(G418) (500 µg/ml) or puromycin (1 µg/ml) (Table1). SAEC proved equally sensitive to either drug, with mostnoninfected cells dying within 24 h. Equal cell numbers weretransferred to 6-well plates, grown to confluence, and allowedto arrest via contact growth inhibition (as measured by propidiumiodide-fluorescence-activated cell sorter analysis) prior toinfection with either ONYX-015 or WT-D adenovirus at a multiplicityof infection (MOI) of 10.

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TABLE 1. Description of p53 mutants used^a

Retroviral expression and infection. ${Phi}$ NX-ampho cells were transfected at roughly 60% confluence withLipofectamine Plus (Invitrogen), and viral supernatant was collectedat 48 and 72 h posttransfection, treated with Polybrene (4 µg/ml),filtered through 0.22-um-pore-size Steriflip filters (MilliporeCorp.), and used directly for SAEC infection. The ${Phi}$ NX-ampho cellmedium was replaced and subsequently collected in a sequentialviral harvest. SAEC cells were incubated with viral supernatant(10 ml in a T150 flask) for 6 h, washed twice, and then returnedto growth in predefined medium. Following a second, repeatedround of retroviral infection 24 h later, 50 to 80% of the cellswere transduced as determined by parallel infection with a greenfluorescent protein-expressing virus and by fluorescence-activatedcell sorter analysis (data not shown).

Characterization of mutant p53-containing SAEC cells. Subconfluent SAEC transduced with either the vector controlor p53 mutants were measured for their ability to incorporatebromodeoxyuridine (BrdU) following etoposide treatment. Cellsin duplicate wells were incubated with etoposide (Sigma) (500ng/ml) for 24 h, washed, and grown in fresh medium for an additional24 h. BrdU (Amersham-Life Science) (10 uM) was added duringthe last 6 h of growth, the cells were fixed with MeOH-acetone(1:1), and the incorporated BrdU was detected using a biotinylatedanti-BrdU antibody (Alexis Biochemicals) and fluorescently labeledstreptavidin (Rockland Inc., Gilbertsville, Pa.). Fluorescentnuclei were counted using a fluorescence microscope (OlympusBX60) and compared with total nuclei (DAPI [4',6'diamidino-2-phenylindole]stained) to determine the percentage of BrdU-positive cells.Four fields were scored from each sample and averaged, and standarddeviations were calculated.

Ionizing radiation and Western blot analysis. Samples for protein analysis were prepared at various timespostinfection. Protein lysates were prepared from subconfluentSAEC transduced with either the vector control or p53 mutantsat 15 h following UV exposure (5 J/m²). Cells were washed threetimes in ice-cold phosphate-buffered saline, scraped in phosphate-bufferedsaline, and pelleted for 5 min at 2,000 x g. Cell pellets wereresuspended in ELB (250 mM NaCl, 50 mM HEPES [pH 7.4], 0.1%NP-40)containing 2 mM dithiothreitol, 5 mM MgCl₂, 1 mM Pefabloc, 1mM EDTA, 25 mM NaF, 1 mM vanadate, and Complete protease inhibitorcocktail (Roche Molecular Biochemicals) and incubated on icefor 30 min. Following centrifugation at 21,000 x g for 10 min,the supernatant was removed and the protein concentration wasdetermined (Bio-Rad protein detection kit). Lysate (30 µgper lane) was loaded on Novex sodium dodecyl sulfate-polyacrylamideelectrophoresis gels and, following electrophoresis, the proteinwas electrophoretically transferred onto a polyvinylidene difluoridemembrane (Immobilon-P; Millipore). The membranes were cut intoseparate molecular-weight ranges and probed as indicated witheither anti-Mdm2 (SMP19; NeoMarkers), anti-p53 (DO-1 and CM-1;gifts from David Lane), or anti-p21 (Transduction Laboratories)antibodies.

Adenoviral infection and viral yield analysis. Contact-inhibited SAEC transduced with either the vector controlor p53 mutants were infected at an MOI of 10 with either WT-Dor ONYX-015 virus. At 48 and 72 h postinfection, cells werescraped in their media (2 ml) and 1 ml of the suspension wastransferred to a 1.5-ml Eppendorf tube. Following three cyclesof freezing and thawing (in a sequence consisting of a dry-ice-EtOHbath followed by a 37°C bath and 10 s of vortex treatment),the sample was centrifuged at 5,000 rpm for 5 min and the supernatantwas collected. The virus concentration of the supernatant wasdetermined in duplicate using HEK293 cells in an enzyme-linkedimmunosorbent assay (ELISA)-based viral quantification assayas previously described (25). Briefly, the viral standard (ONYX-015)and unknown samples were serially diluted and used to infectHEK293 cells in a 96-well plate. At various times postinfection,the medium was removed, the cells and virus were fixed, andviral particles were detected using anti-adenoviral antibodiesand an ELISA-based detection system. The viral yields shownrepresent the peak yields of triplicate averages from eitherthe 48- or the 72-h time point. Typically, this represents the48-h time point for WT-D-infected and the 72-h time point forONYX-015-infected cells.

RESULTS

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Results

To evaluate the role of p53 function in determining ONYX-015replication, we used early-passage primary SAEC as a model system.These cells were chosen for a number of reasons. First, theyrepresent a physiologically relevant target cell of human adenovirus.Second, they are highly infectible, with more than 95% of cellsinfected at an MOI of 10 PFU/cell (data not shown). Third, SAECare isolated from normal human donors and are genetically wildtype and therefore presumed to contain intact afferent and efferentp53 pathways. Last, as described below, the use of SAEC offersthe ability to compare multiple different p53 mutant allelesin a genetically matched, nonclonal cell population.

As determined by viral yield assay, SAEC enable selective replicationof WT-D (wild-type control adenovirus) compared to ONYX-015virus, particularly under conditions of contact inhibition andgrowth arrest (Fig. 1A). This result resembles a previouslyreported observation by Goodrum and Ornelles (16), who showedthat the yield of E1B-55K-deficient virus was inversely relatedto the plating density at which HeLa cells were infected. Onthe basis of this observation, we performed all subsequent infectionsand viral yield analyses under conditions of contact arrest(S phase < 1%; data not shown). Interestingly, despite thevirus's attenuated level of replication, ONYX-015 induced amorphological change similar to CPE in contact-arrested SAECcomparable to that induced by WT-D (Fig. 1B). Thus, CPE is notsimply a function of viral replication and cannot be used asa readout for productive viral infection.

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FIG. 1. Characterization of SAEC. (A) SAEC were infected at a range of cell plating densities with either WT-D or ONYX-015 virus, and viral yields were determined. Results of viral yield levels determined by ELISA assay (see Materials and Methods) at 72 postinfection are shown as the ratio of WT-D viral yield to that of ONYX-015. Error bars indicate standard deviations. (B) Assessment of CPE in contact-arrested SAEC at various times following infection with either WT-D or ONYX-015.

To abolish p53 function in SAEC, we exogenously expressed avariety of mutant p53 genes. The mutants chosen representedall three p53 mutant categories: mutants 143A and 175H are examplesof class II or conformational mutants in which p53 activityhas been disrupted by a global change in tertiary structure;248W is representative of a class I or contact site mutant inwhich p53 residues directly involved in protein-DNA contactsites have been altered and yet protein conformation remainsintact; and, finally, GSE is a 94-amino-acid polypeptide derivedfrom a region of p53 that includes the C-terminal oligomerizationdomain (38). Genes expressing the various p53 mutants or theappropriate vector controls (Table 1) were introduced into SAECby means of amphotropic retrovirus delivery (Fig. 2; also seeMaterials and Methods), and drug-resistant cells were obtained.High levels of p53 were detected in all cells expressing mutantversions of p53 but not in vector-only control cells (Fig. 3b,-UV lanes). The elevated levels of full-length p53 observedin GSE-expressing cells represent endogenous, wild-type p53,whose expression is presumably increased in response to lossof the Mdm2-dependent negative-feedback loop (29).

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FIG. 2. Outline of the experimental approach in early-passage (2) SAECs (Clonetics Corp.). See Materials and Methods for details.

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FIG. 3. Characterization of SAEC containing mutant p53 or vector control. (A) Percentages of BrdU-positive cells following etoposide treatment compared to percentages of control (untreated) cells. Error bars indicate standard deviations. (B) Response of cells to UV irradiation. The results of immunoblot analysis of p53, GSE, Mdm2, and p21 expression at various times following exposure to UV at 5 J/m² (+) or mock treatment (-) are indicated. Molecular mass markers are indicated.

Following selection, the cells were characterized to assesstheir p53 function. We chose two well-characterized p53 assays:cell cycle arrest in response to double-stranded DNA breaksand induction of p53 target genes in response to UV irradiation(Fig. 3). Subconfluent SAEC transduced with either the vectorcontrol or p53 mutants were treated with the topoisomerase IIinhibitor etoposide and assayed for their ability to arrestcell cycle entry, as determined by BrdU incorporation. All ofthe mutant p53 constructs examined abolished this checkpoint,albeit to various degrees (Fig. 3A), indicating that p53 functionwas indeed compromised in these cells. In contrast, the etoposidecheckpoint was intact in both nontransduced cells (data notshown) and vector-only control cells (Fig. 3A, lanes LXSN andpBabe), as evidenced by the presence of little or no BrdU incorporation.

To further characterize p53 function, cells transduced witheither the vector control or p53 mutants were UV irradiatedand cell lysates were then examined by Western blot analysisfor the expression of p53 target genes (Fig. 3B). Interestingly,the increased level of p53 protein seen in some cell types inresponse to UV irradiation was not seen in SAEC. This is consistentwith recent reports showing attenuated increases in p53 levelsfollowing DNA damage in primary epithelial cells (5, 10, 32)for reasons that are presently unclear. Increases were noted,however, in the level of the p53 target genes Mdm2 and, to amodest extent, p21 following UV irradiation (Fig. 3B; compare+ versus - lanes and LXSN versus pBabe lanes). Notably, theseincreases were attenuated to various degrees in SAEC expressingthe mutant p53 genes, thereby indicating elimination of p53function in certain cases. The GSE mutant appeared to be mosteffective in blunting p53's transcriptional response to UV (Fig.3B and data not shown), and 143A appeared to have the weakesteffect in both assays. Thus, by each of these two criteria,SAEC expressing the various mutant p53 alleles were demonstratedto have altered and attenuated p53 function and, as such, weresuitable for use in evaluating the role of p53 in replicationof a ${Delta}$ E1B 55kD adenovirus.

To measure adenoviral replication in these cells, pooled, drug-resistantpopulations of retrovirally infected SAECs were plated on six-welldishes, grown to confluence, and allowed to arrest due to prolongedcontact inhibition. As previously shown (Fig. 1A), contact-dependentgrowth arrest was required to obtain strong, selective decreasein ONYX-015 virus replication compared to that seen with WT-Dvirus in human primary SAECs. Prior to adenoviral infection,a sample of the cells was harvested and assessed by propidiumiodide staining for cell cycle analysis. Both the vector controland mutant p53-containing cells were growth arrested with atleast 90% of cells in G₀/G₁ (data not shown). Cells in duplicatewells were infected with either WT-D or ONYX-015 adenovirusat an MOI of 10 (see Materials and Methods), and viral yieldswere determined at either 48 or 72 h postinfection (Fig. 4A).The ratio of the peak WT-D viral yield to that of ONYX-015 wascompared to provide an estimate of the relative degree of viralreplication (Fig. 4B). As expected, both vector control samplesshowed an approximate 30-fold-enhanced replication of WT-D relativeto ONYX-015 (Fig. 4B). Interestingly, expression of mutant p53affected ONYX-015 replication to various degrees and dependedupon the specific p53 mutant that was examined. Cells that expressedGSE and 175H (p53 mutants that each showed strong evidence ofblocking p53 activity in functional assays) (Fig. 3), showedonly a moderate affect in rescuing ONYX-015 replication (Fig.4). This suggests that loss of p53 function alone is not sufficientto restore the level of replication of E1B 55kD-deficient adenovirusto that of WT-D. In contrast, cells expressing the 248W p53mutant were unique in showing a marked increase in ONYX-015replication compared with vector-only control cells, yieldingan average viral yield ratio of 3.5 (Fig. 4B). The fact thatthis mutant was unique in its ability to rescue E1B 55kD-deficientviral replication suggests that some other activity of the mutantbeyond simple elimination of endogenous wild-type p53 functionwas required to restore viral replication. Surprisingly, cellsexpressing the p53 mutant 143A showed increased discriminationbetween WT-D and ONYX-015; replication of the E1B 55kD-deficientadenovirus was more attenuated in these cells than in vector-onlycontrol cells. We postulate that this result may be due to partialwild-type activity of this temperature-sensitive p53 mutant(see Discussion).

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FIG. 4. Viral yield in SAEC containing mutant p53 or vector control. (A) Viral yield for either ONYX-015 or WT-D virus measured at 72 h from a representative experiment. The light and dark bars represent ONYX-015 and WT-D, respectively. The values represent average viral yield measurements determined in triplicate from duplicate experiments. Units indicated are the equivalent of PFU/ml (see Materials and Methods). Error bars indicate standard deviation. (B) Ratio of WT-D to ONYX-015 average peak viral yields. The values shown represent the peak yields from either the 48- or 72-h time point. In some cases this represents the 48-h time point of WT-D-infected and the 72-h time point of ONYX-015-infected cells. Results represent data from two independent experiments, and standard deviations are indicated.

DISCUSSION

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Discussion

In understanding human cancer, one theme that is now clear isthat disabling a cell's p53 function is a critical and nearlyuniversal event in tumorigenesis. In this study, a genetic meanswas used to selectively abolish p53 function. This approachprovides the ability to create multiple matched experimentaland control cell populations to investigate the contributionof a single gene product in an otherwise wild-type cell. Inthis case, p53 function was altered in cells representing thephysiologic target for a novel therapeutic adenovirus, leadingto several interesting and important findings.

We determined that loss of p53 function alone was not sufficientto allow ONYX-015 virus replication. This is consistent withseveral recent reports and is in contradiction to ONYX-015'spurported mechanism of action. In cell populations expressingeither of two strong dominant-negative p53 mutants, GSE and175H, almost no effect was seen on ONYX-015 replication in comparisonto that of control cells. Although loss of p53 function wasdetermined by a variety of assays, it is possible that suchmutants did not inhibit all aspects of p53 function and thatthe component of p53 action required for viral attenuation remainedintact. Since we do not currently understand p53's exact mechanismin this regard, we cannot rule this out as a possibility. Anumber of observations, however, suggest that this is unlikely.Structural characterization of p53 and the use of conformation-specificimmunological probes indicate that mutations at residue 175alter protein conformation and disrupt sequence-specific DNAbinding (39), generally considered the cardinal function ofp53. Similarly, GSE assembles with p53 to form a hetero-oligomericcomplex that is unable to bind DNA. Furthermore, one of thesemutants, 175H, not only arises as one of the most frequent p53mutations in human tumors (18) but has now been shown to predisposemice to malignancies when expressed in germ line cells (30).Mutation at residue 175, thus, appears sufficient to abolishcritical p53 functions required for tumor suppression.

In this study, one p53 allele, 248W, acted to enhance ONYX-015virus replication. In this regard, we propose the existenceof a gain-of-function role for this allele. There is precedencefor gain of function in p53 mutants. Specifically, in assayssuch as growth in soft agar and oncogenic transformation, expressionof certain p53 mutant alleles in a p53 null background showevidence of higher levels of tumorigenicity than cells of thenull background alone (7, 9, 49). In an observation that mayaccount for some of these findings, it has been shown that certainp53 mutants that disrupt conformation (class II) demonstrategain of function in eliminating a cell cycle spindle checkpoint(19). Moreover, certain p53 mutants have the ability to bindto and disrupt the function of p73, a p53-related protein (6,11, 31), thereby providing another possible gain-of-functionmechanism. Further evidence comes from recent work that suggestsa gain-of-function role for mutant p53 in promoting androgen-independentgrowth of prostate cancer cells in culture (35). In the caseof 248W, the mechanism of action in enhancing E1B 55kD-deficientviral replication is not clear. Importantly, residue 248 isthe most frequent site of p53 mutation in human cancers. Amongthe p53 mutants assayed in this report, 248W is unique in thatit is a contact site (class I) mutant. Mutants of this classare believed to retain an overall wild-type structural conformationbut harbor point mutations at specific residues required forsite-specific DNA binding. In the case of adenoviral replication,a mutant of this type may act to override a cellular checkpointthat inhibits viral replication by binding and sequestrationof p53 conformation-dependent protein partners that negativelyregulate viral replication. Specifically, another class I mutant(273H) has been shown to constitutively interact with topoisomeraseI (15) and promote gene amplification in Saos-2 cells (8). Alternatively,a mutant allele such as this may provide more favorable conditionsfor viral replication by relocalizing p53 to specific subcellularcompartments or molecular complexes that enhance viral replicationor by inducing additional cellular components, such as molecularchaperones, that facilitate viral biogenesis (14).

Even 248W, the mutant p53 allele with the greatest ability torescue viral replication, did not increase the level of replicationof ONYX-015 virus to that of wild-type adenovirus. Given theknown requirement for the E1B 55-kDa protein in other viralprocesses, such as the regulation of RNA export and translation,it is not surprising that complete rescue was not observed.Surprisingly, 143A expression led to an apparent increased discriminationbetween WT-D virus and ONYX-015. This effect was due to an increasein WT-D viral yield as well as a decrease in that of ONYX-015(Fig. 4A). One possible explanation may be the partial wild-typephenotype that this particular p53 allele is known to demonstrate,since it can alternate between mutant and wild-type conformationsin a temperature-dependent manner. We speculate that high levelsof p53 with partial wild-type function may result in a superinductionof p53 activity.

ONYX-015 shows clinical promise as a novel agent in human tumortherapy and is now being evaluated in phase III clinical trialsof head and neck cancer. As such, its molecular mechanism ofaction is of obvious interest. Similarly, an understanding ofspecific genetic characteristics that promote ONYX-015 replicationwould provide a valuable means to guide clinical intervention.Within the limited p53 sequence data available from phase IIONYX-015 trials, there are a few (3 of 22) examples of p53 mutationsof the class I type. These are currently being evaluated forcorrelation with clinical outcome.

ACKNOWLEDGMENTS

We are grateful to Valeria Ossovskaya and Martin Oft for helpfulsuggestions and advice and to Leisa Johnson and Michael Kornfor valuable comments on the manuscript. We thank members ofthe Lane (University of Dundee) and Tlsty (UCSF) laboratoriesfor p53 antibodies and plasmid constructs.

FOOTNOTES

* Corresponding author. Mailing address: UCSF Mt. Zion Cancer Center, Box 0875, San Francisco, CA 94143-0875. Phone: (415) 502-6791. Fax: (415) 502-6779. E-mail: bhann{at}cc.ucsf.edu.

REFERENCES

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