[CANCER RESEARCH 59, 1391–1399, April 1, 1999]

Review

Apoptosis, p53, and Tumor Cell Sensitivity to Anticancer Agents1

J. Martin Brown 2 and Bradly G. WoutersCancer Biology Research Laboratory, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305

Abstract

A widely held tenet of present day oncology is that tumor cells treatedwith anticancer agents die from apoptosis, and that cells resistant toapoptosis are resistant to cancer treatment. We suggest, in this review,that this tenet may need to be reexamined for human tumors of nonhe-matological origin, for two principal reasons: (a) cell killing has often beenassessed in short term assays that are more influenced by the rate, thanthe overall level, of cell killing. This has tended to underestimate cellkilling for cells not susceptible to apoptosis or having mutantp53; and (b)conclusions from experiments with normal cells transformed with domi-nant oncogenes have often been extrapolated to tumor cells. This does nottake into account the fact that tumor cells have invariably undergoneselection to an apoptotically resistant phenotype. In this review, we exam-ine the impact of these two factors with particular emphasis on theinfluence of mutations in p53 on the sensitivity of tumor cells to DNA-damaging agents. We find that because wild-typep53predisposes cells toa more rapid rate of cell death after DNA damage, particularly withnormal or minimally transformed cells, that short-term assays have led tothe conclusion that mutations inp53confer resistance to genotoxic agents.On the other hand, if clonogenic survival is used to assess killing in cellsderived from actual solid human tumors, then apoptosis and the genescontrolling it, such as p53and bcl-2, appear to play little or no role in thesensitivity of these cells to killing by anticancer drugs and radiation.

Introduction

During the past 10 years, interest of basic scientists and cliniciansin the influence of programmed cell death, or apoptosis, on thesensitivity of tumors to anticancer treatment has risen and continues torise dramatically. A major reason for this interest is that apoptosis isa defined program of cell death that is markedly influenced bothpositively and negatively by a variety of genes, many of which aremutated and/or dysfunctionally regulated in human cancers (1).Among the most important of these are the tumor suppressor genep53and members of thebcl-2 gene family (1, 2). The fact that apoptosisis a genetically defined pathway has led to two principal expectations:(a) that the genotype of the tumor will be predictive of the outcome ofcurrent anticancer therapy; and (b) that new therapies based on apop-tosis will be superior to present-day anticancer treatments. The re-quirement for wild-typep53 for apoptosis after genotoxic damagecaused by anticancer agents including irradiation has been well dem-onstrated, particularly in oncogenically transformed rodent cells andin tissues of lymphoid origin (3, 4). However, the influence ofp53andother genes on apoptosis in malignant tissues of nonhematologicalorigin is by no means clear. There have also been reports indicatingthat apoptosis does not correlate with the total cell kill measured byother means following anticancer therapies. In this review, we will

focus on tumor cells of nonhematological origin. In particular, wereview critically the data underlying the hypothesis that these cancercells when treated with radiation or chemotherapeutic drugs die ofapoptosis, and that cells resistant to apoptosis are resistant to cell killby anticancer therapy.

Many genes have been identified that affect the extent to whichcertain cell types undergo apoptosis during normal development andafter pathological stress. Together with the assumption that apoptosisplays a major role in cell killing by DNA-damaging agents, thesegenetic studies have led to the present hypothesis that tumors withmutations inp53, high levels of bcl-2, or high ratios of bcl-2:baxshould be resistant to cancer treatment (1, 2, 5). Because there is nowa wealth of data from clinical studies in which outcome has beencorrelated with the status of these and other genes affecting apoptosis,this hypothesis would seem an easy one to test. However, a majorproblem with such analyses is that it is often impossible to separatetreatment sensitivity from patient prognosis. For example, tumorswith mutatedp53 can be more anaplastic, can have a higher propor-tion of proliferating cells, can be more metastatic, and in general canhave a more aggressive phenotype that similar tumors with wild-typep53(6). This can lead to a worse prognosis for patients whose tumorshave mutatedp53 independent of treatment sensitivity (7). Havingsaid this, there are numerous examples in the literature wherep53mutations (or high levels of p53 protein by immunohistochemistry)either do not affect patient prognosis (8, 9) or lead to better outcomeafter treatment (10, 11). In a comprehensive review of the clinicalsignificance ofp53mutations in human tumors, Bosari and Viale (12)concluded (in 1995) that a definite answer could not yet be given tothe question or whetherp53 aberrations led to a more aggressivephenotype or to treatment resistance.

Apoptosis and Sensitivity to Anticancer Therapy: The PresentView. As we point out above, because mutations inp53or other genesmay affect tumor aggressiveness and patient prognosis, it is difficultto obtain from clinical data an answer to the question of the role ofp53 or of apoptosis in treatment sensitivity. However, experimentalsystems can be not only free of such biases, they can also use moderngene knockout, transgene, and other molecular techniques to answerthe narrower question of: “Does the level of apoptosis and/or genescontrolling apoptosis affect the sensitivity of cancer cells to killing bygenotoxic agents?”

The present view is that this is the case (1, 2, 5, 13, 14). It hasbecome widely accepted that cell death after DNA damage by anti-cancer agents is primarily the result of apoptosis, and that cells thatcan evade apoptosis will be resistant to cell killing. Often cited for thisview, and in particular the role of mutatedp53 in radiation andanticancer drug resistance, are pioneering studies with dominant on-cogene-transformed normal fibroblasts from embryos ofp53 wild-type (p531/1) andp53 knockout mice (p532/2) (15, 16), as well ashighly significant associations of mutatedp53with drug resistance inthe National Cancer Institute panel of 60 cell lines used for screeningnovel potential anticancer drugs (17).

However, despite the seemingly strong case that cells die fromcancer treatment due to apoptosis largely controlled by wild-type p53,

Received 11/25/98; accepted 2/2/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by United States National Cancer Institute Grant CA 15201(to J. M. B.) and by a National Cancer Institute of Canada Research Fellowship(to B. G. W.).

2 To whom requests for reprints should be addressed, Cancer Biology ResearchLaboratory, GK103, Department of Radiation Oncology, Stanford University School ofMedicine, Stanford, CA 94305-5468.

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several investigators have reported results that contradict this hypoth-esis, particularly when they have measured both apoptosis and overallcell killing by a colony-forming assay. For example, it has beenreported that large changes in apoptosis do not lead to any changes ineventual cell killing (18–23), or that the status ofp53does not affectsensitivity to DNA-damaging agents. However, the present confusioncan be largely resolved if two facts are borne in mind.

(a) Many investigators have used methods for assessing the extentof cell killing by anticancer drugs and radiation based on earlyfunctional changes (such as dye uptake), or on growth inhibition,rather than on colony formation. These assays can lead to incorrectassessments of overall cell kill, largely because they ignore kineticdifferences in the manifestation of cell death.

(b) Conclusions derived from normal cells transformed with dom-inant oncogenes such asE1Aandmychave been extrapolated to tumorcells. However, apoptosis, particularly the early apoptosis character-istic of cells of lymphoid origin and oncogene-transformed normalcells, is often an insignificant mode of cell death for the cells of themajority of solid tumors, irrespective of the status of genes such asp53 andbcl-2.

Both of these issues are explored in more depth in the followingsections.

Short-Term Assays Can Underestimate Overall Cell Killing.How should cell killing be measured after a toxic insult? This wouldnot seem to be a particularly difficult problem; a dead cell has manydistinct morphological features, loses metabolic functions, and fails toexclude dyes such as propidium iodide and trypan blue. If death is dueto apoptosis, then a number of well-characterized features occurincluding chromatin condensation and fragmentation, formation ofnucleosomal “DNA ladders,” and exposure of phosphatidyl serine inthe outer cell membrane that can be detected with annexin V. Thus,dead cells can be readily identified, and their proportion in a popula-tion readily quantitated. However, identification of dead cells oncethey have died is not the problem. The problem is that cells do not dieimmediately after treatment; they can take hours to many days beforedying, and this is highly dependent upon the cell type and the toxicagent being investigated (19, 24–26). Despite this, many investigatorsassessing cellular sensitivity to genotoxic agents measure viability by

total population staining [the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide or XTT3 assays] or assess the extent of celldeath from the proportion of cells incorporating a dye excluded bylive cells, such as trypan blue or propidium iodide, at short times (1–4days) after treatment. As we point out below, this can lead to anunderestimate of the overall level of cell killing.

The problem of correctly assessing the fraction of cells surviving agiven treatment was solved for mammalian cells in the mid-1950s byPuck and Marcus (27), who developed the technique of cloningindividual cells in vitro. The ability of a single cell to grow into acolony (usually defined as.50 cells) is an assay that tests every cellin the population for its ability to undergo unlimited division. It is, formammalian cells, the exact counterpart of assays measuring bacterialor yeast survival after treatment with cytotoxic agents. The assay hasbecome widely used for assessing the response to cytotoxic agents ofcells in vitro, as well as cells in normal tissues and tumorsin vivo.When the logarithm of the percentage of surviving cells determinedby the clonogenic assay is plotted against the dose of agent used, astraight line (sometimes with an initial “shoulder” region) is usuallyobtained, implying an exponential relationship between dose and cellkill. Both for radiation and anticancer drugs, this “log cell kill”hypothesis for cell survival has been demonstrated under many dif-ferent conditions to be effective in predicting the radiation or chem-otherapy dose needed to cure experimental mouse tumors or multi-cellular spheroids (28–30).

Fig. 1 shows data illustrating the problem of using short-termassays to assess the influence ofp53 on the sensitivity of cells toanticancer therapy. In this experiment, MEFs from wild-type(p531/1) or p53 knockout mice (p532/2) and transformed withE1Aandras (15) were exposed to different concentrations of etoposide for1 h and assayed either by the XTT assay or by clonogenic survival.The oncogene-transformed p531/1 are very sensitive to apoptosis andstart to die 3–6 h after treatment, whereas the p53-/- cells die muchlater (1–2 days after exposure). Thus, viability measured at 1 day aftertreatment, as in the XTT assay above, markedly underestimates even-tual cell killing in the apoptotically insensitive cells.

Short-term assays can also be a problemin vivoas illustrated in Fig.2. Here we have grown theE1A- and ras-transformed MEFs fromwild-type (p531/1) and p53 knockout mice (p532/2) as tumors insevere combined immunodeficient mice and measured their responseto a single dose of irradiation. Because the p531/1 cells undergo earlyapoptosis, leading to rapid and extensive tumor shrinkage, an assess-ment of tumor response by measuring tumor size within 2 weeks ofirradiation would conclude that the p531/1 tumors were more radio-sensitive. This is because the rate and/or extent of tumor shrinkage islargely governed by the rate of cell death rather than by the overalllevel of cell kill. However, growth delay, usually to 2–3 times treat-ment volume, is a way of assessing tumor response that is not affectedby the rate of cell death (31, 32) and, as can be seen in Fig. 2, suggeststhat the initial apoptotic response observed in the p53 wild-typetumors has little or no effect on the final growth delay of these tumors.

The ability of cells to undergo unlimited proliferation as tested bytheir ability to form a colony has, therefore, become the “gold stand-ard” for assessment of cellular sensitivity to cytotoxic treatments. It is,however, important to be precise about the meaning of this; it is thebest way to assess the proportion of cells surviving a particulartreatment under the experimental conditions used. It does not meanthat all responses of tumors can be reliably predicted using a clono-genic assay of the cells treatedin vitro. This was recently illustratedin studies of Waldman and colleagues and by us (22, 23). HCT116

3 The abbreviations used are: XTT, 2,3-bis[2-methyl-4-nitro-5-sulfophenyl]-2H-tetra-zolium-5-carboxanilide inner salt; MEF, mouse embryo fibroblast.

Fig. 1. Short-term assays can wrongly assess sensitivity of cells to anticancer agents.MEFs from wild-type (p531/1) or p53 knockout mice (p532/2) and transformed withE1A and ras were exposed to different concentrations of etoposide for 1 h and assayedeither by the XTT assay 1 day after exposure or by clonogenic survival after 8 days ofincubation. Pooled data from two independent experiments are shown; bars, SE.

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human colon carcinoma cells isogenic for the p53 target genep21waf1

(designated p211/1 or p212/2) had the same response when tested byclonogenic survivalin vitro (although they differed greatly in theirlevel of apoptosis under the same conditions). However, when tumorswere grownin vivo from the same cells, those from p212/2 cells weremuch more sensitive to radiation as measured by growth delay thantheir wild-type counterpart. This was originally suggested as a failureof the clonogenic assay to correctly reflect tumor cell sensitivity (22,33, 34). However, if clonogenic survival is assessed from the irradi-ated tumors, then there is close agreement between this assay and thein vivo growth delay assay for the two cell lines (23). The difference,therefore, between thein vitro and in vivo data are the result ofdifferent environmental conditions of the cells at the time of treat-ment.

When used with care, the clonogenic assay is probably the mostreliable method for assessing cell killing after genotoxic agents (35).With this in mind, we can now ask the question of whether apoptosisis a reliable determinant of cell killing to radiation and anticancerdrugs when assayed by clonogenic survival. Although there are manyexamples in the literature, particularly for cells of hematologicalorigin, where apoptosis and cell killing assayed by clonogenic sur-vival are well correlated, there are also many examples, particularlyfor cells of nonhematological origin, where this is not the case. Fig. 3,for example, shows some of our own results in which the response ofHCT 116 cells that are either p211/1 or p212/2 have been assessedboth for apoptosis and for clonogenic survival under identical treat-ment conditions to radiation, to etoposide, and to the bioreductivedrug tirapazamine. It is clear from these data, and from other exam-ples in the literature (18–20, 36), that the extent of apoptosis is not areliable indicator of cellular sensitivity to anticancer agents. Themessage from Fig. 3 is that cells can die from apoptosis at differentrates, and that the dominant mode of cell death may, or may not be,apoptosis.

The same conclusion is also likely to hold for tumorsin vivo. Asnoted above, the HCT116 p212/2 colon carcinoma cells are sensitiveto radiation when growing as tumorsin vivo. This is associated witha massive extent of apoptosis after irradiation (.90% of the tumorcells as determined by the terminal deoxynucleotidyltransferase-me-

diated nick end labeling assay).4 This apoptosis can be almost com-pletely eliminated by overexpressing Bcl-2 in these cells. Surpris-ingly, however, this does not affect the response of the tumors toradiationin vivo.4

Of note from the data in Figs. 1 and 3 is that the sensitivity of cellsto killing as assessed by their ability to form a colony is greater thanthat for the short-term assay that measures the proportion of cellsundergoing apoptosis. In other words, for a given toxic insult, morecells are killed when assessed by clonogenic assay than undergoapoptosis. This has been noted previously by several investigators (19,37, 38), but it is contrary to the widely held view that the traditionalmode of cell killing by radiation and anticancer drugs requires agreater amount of DNA damage than is required for apoptotic death.However, it is important to be aware of the fact that it can be difficultin some circumstances, particularly for tissues and tumorsin vivo,to assess apoptosis, because it is a dynamic process and not all ofthe cells destined to die by apoptosis may be visible at any one time(37, 39).

These data lead to the conclusion that the response of tumor cellseither in vitro or in vivo to genotoxic damage may, or may not,correlate with the extent of apoptosis after exposure. In the nextsection, we examine one particular aspect of genotype and sensitivity,i.e., the impact of wild-type or mutantp53 on cellular sensitivity togenotoxic damage.

Does p53 Status Affect the Sensitivity of Cells of Nonhemato-logical Origin to Genotoxic Damage?Although there are manystudies of the influence ofp53 status on the sensitivity of cells togenotoxic agents, in view of the above discussion concluding thatshort-term assays can be misleading, we have considered only thosestudies that have used clonogenic survival (which is not affected bythe rate of cell death). We have also focused primarily on thoseinvestigations using ionizing radiation. This is because different an-ticancer drugs have different mechanisms of action that could involvep53 directly independent of apoptosis (e.g., involving nucleotideexcision repair), and because investigators use a wide variety ofexposure conditions for drugs but use much more defined radiationexposure conditions. There are also more studies with ionizing radi-ation than with all anticancer drugs combined.

In reviewing the literature on the question of the influencep53 onthe radiation response of cells, we have therefore applied the follow-ing criteria:

(a) Only cells or tissues of nonhematological origin have beenconsidered.

(b) Only investigations in which clonogenic survival was used toassess cell killing were considered.

We have also excluded from the analysis studies in which wild-typep53 was massively overexpressed in cells using viral vectors beforeirradiation. Typically, this results in radiation-induced apoptosis andradiation sensitization (40, 41).

The data in the literature conforming to these criteria fall into twomain categories: (a) First are those papers in which a group ofnongenetically matched cell lines have been assessed for statisticaldifferences in radiation sensitivity between those that have wild-typep53and those that have mutantp53. Of the 27 publications in the totalpool, 10 fall into this category, of which 3 findp53mutated cells moreradioresistant (42–44), 3 findp53 mutations make no difference toradiation sensitivity (45–47), and 4 findp53 mutated cells moreradiation sensitive (48–51). Illustrative data from the two largestpublished series, each assessing radiation sensitivity andp53 status

4 B. G. Wouters and J. M. Brown, unpublished data.

Fig. 2. Short-term assays of tumor response can incorrectly assess overall cell kill fortumors whose cells have different probabilities of undergoing early apoptosis. MEFs fromwild-type (p531/1) or p53knockout mice (p532/2) and transformed withE1A and raswere grown as tumors in severe combined immunodeficient mice and irradiated with 15Gy of irradiation at time 0. Tumor volume was assessed three to five times weekly bymultiplying three perpendicular tumor diameters. The tumors were irradiated (or enteredinto the control groups) when they reached a mean diameter of 5–7 mm. Shown are thegeometric means of the volumes of five mice per group relative to the volumes at time 0.Growth delay relative to controls at two times treatment volume is shown.

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in cell lines or primary cultures from human cancers, are shown inFig. 4.

The second category includes publications in which investigatorshave used genetically matched cell linesin vitro or tissuesin vivofrom p531/1 versusp532/2 mice, or have used cells with or withoutexpression of the HPV protein E6, or have used transfection of mutantp53or conditionally expressedp53. In this group, five investigationsfoundp53null/mutated cells more radioresistant (52–56), nine foundno difference in radiation sensitivity betweenp53 wild-type or null/mutant cells (55, 57–64), and three foundp53 null or mutant cellsmore sensitive that wild-type cells (59, 65, 66). It should be noted herethat three of the five studies showing that loss of p53 function resultsin radioresistance used overexpression of mutant p53 in ap53 wild-type background (52–54). In a recent reanalysis of the largest of thesestudies, the resistance of the mutant p53-expressing cells was corre-lated with more proficient rejoining of DNA double-strand breaks(67). However, there was considerable overlap in sensitivity betweenthe wild-type and mutant-expressing cells, and significant resistancedid not occur until mutant p53 was overexpressed more than 100-fold.

Clearly, these data do not justify the conclusion thatp53mutated ornull cells are in general more resistant to radiation-induced cell killthan arep53 wild-type cells.

For anticancer drugs, there have been surprisingly few studies usingclonogenic survival to determine the influence ofp53 on the sensi-tivity of cells of nonhematological origin. However, when this assayhas been used, loss of wild-typep53has been found to have no effecton the sensitivity of cells to the topoisomerase I inhibitor, campto-thecin (61), to doxorubicin (19, 68), or to taxol or vincristine (68).Interestingly, mutations inp53have been shown to confer sensitivityto drugs whose toxicity is modulated by nucleotide excision repair,such as nitrogen mustard and cisplatin (38, 68). The study with murineteratocarcinoma cells is of particular interest because the absence ofp53 protected against cisplatin-induced apoptosis, yet sensitized to

cell kill determined by colony formation (38). This can be readilyunderstood in terms of the drug doses used; to reach 90% cell kill byapoptosis in the most apoptotically sensitive cell line required some50 times more cisplatin exposure than to produce an equal level of cellkill by clonogenic assay in the same cell line.

Given the above evidence thatp53status does not generally affectthe sensitivity of cells to radiation and anticancer drugs, it is pertinentto ask how such a belief arose. Several factors have probably con-tributed.

(a) Lymphocytes, thymocytes, and lymphoma cells, are highlysensitive to anticancer therapy and invariably die a rapid apoptoticdeath that is dependent on wild-typep53 (3, 69).

(b) Short-term assays, rather than clonogenic survival, have oftenbeen used to assess cell killing. These can be markedly affected by therate at which cells die, which, in turn, can be dependent on the modeof cell death (e.g.,apoptosis or necrosis), and on the cellular genotype.

(c) As mentioned earlier, conclusions derived from normal cellstransformed with dominant oncogenes have been extrapolated totumor cells. We point out in the following section that these trans-formed normal cells are hypersensitive both to apoptosis and to killingas measured by clonogenic survival. The hypersensitive phenotypelikely results from a synergy between oncogene activation of p53 andthe DNA damage response and is not the case for tumor cells ingeneral.

(d) The highly significant correlation of mutatedp53 with resist-ance to active anticancer agents (that act principally by damagingDNA) in the National Cancer Institute screen of 60 different cell lines(17) is actually a correlation, not of cell kill, but of 2-day growthinhibition, with p53 status. Because wild-typep53 is required forgrowth arrest after DNA damage (70), such a correlation of sensitivityto growth inhibition with wild-typep53 is expected. Because there islittle or no correlation of growth inhibition with cytotoxicity across

Fig. 3. Apoptosis does not necessarily predict overallsensitivity to genotoxic agents. The response of HCT116cell lines isogenic for the CDK inhibitorp21waf1 are shownfollowing three different genotoxic treatments: etoposide,radiation, and tirapazamine under hypoxia. The upperframes show that the loss of p21 (solid lines) sensitizes cellsto death by apoptosis for each of these treatments in com-parison with the wild-type cell line (dashed lines). How-ever, the overall sensitivity as assessed by clonogenic assay(lower frames) is unchanged.Upper panels,the apoptoticfraction following etoposide (5mg/ml), irradiation (10 Gy),and tirapazamine (20mM) under hypoxia. The data foretoposide and irradiation are redrawn from Wouterset al.(23). Bars,SE.

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different cell lines (71), these data do not provide evidence for theinvolvement ofp53 in drug sensitivity to cell killing.

In summary, although wild-typep53 can influence the decision ofwhether cells undergo apoptosis after genotoxic insult, and, impor-tantly, this leads to differences in the rate at which cells die, theavailable data do not support a role for p53 in determining the overalllevel of cell kill in tumor cells.

Having said this, there may be subtle influences ofp53abrogationon sensitivity to radiation or some anticancer drugs in some cellstypes. For example, Yountet al.(55) showed that although abrogatingp53 function did not change the overall radiosensitivity of humanglioblastoma cells, it did increase the resistance of synchronized cellswhen irradiated in early G1. It also appears that mutantp53, throughits inability to transactivatep21, increases the sensitivity of chemo-therapeutic agents, such as cisplatin and nitrogen mustard, that requirenucleotide excision repair in their repair pathway (68).

Are There Situations in Which Apoptosis Can Contribute toSensitivity to Cytotoxic Agents? Apoptosis is a major form of celldeath that is dependent on wild-type p53 after DNA damage forcertain normal cells, such as those of the early embryo and those oflymphoid origin (3, 4). There are also several examples in the litera-ture where apoptosis clearly contributes to the overall sensitivity ofcells to treatment with radiation or chemotherapeutic agents as as-sessed either byin vivo treatments (15, 72) or by clonogenic assaysinvitro (69, 72, 73). However, a major difference between these studiesand ones that have failed to find a link between apoptosis and overallsensitivity has been the choice of the cells used in the study. For cellsof nonlymphoid origin, the cases in which apoptosis does contributeto overall sensitivity have in large part been those that used cellsderived from normal tissues genetically engineered to express domi-nant oncogenes. Introduction of viral or cellular oncogenes such asE1A (74, 75),myc(72, 76), or human papillomavirusE7 (77) renderscells dramatically more sensitive to apoptosis in response to variousgenotoxic or nongenotoxic stresses and can also make these cellshypersensitive to overall killing by radiation (15, 78) and variouschemotherapeutic agents.5 Apoptosis in these cases is characteristic of

lymphoid cells: death is usually rapid after treatment, is dependent onthe status ofp53 (16, 79), and can be inhibited by overexpression ofbcl-2 (72, 80–82). For example, in MEFs transformed withE1AandHa-ras, cells with wild-typep53undergo rapid apoptosis in responseto genotoxic and nongenotoxic stress, whereas similarly transformedcells fromp53 knockout mice do not (16, 82). This dramatic differ-ence in apoptosis also translates into an increased sensitivity of thetransformed p531/1 MEFs in terms of the overall sensitivity asdetermined either by clonogenic survival5 or by tumor responseinvivo (15).

The recent discovery of ARF, a protein encoded by an alternativereading frame within theINK4a locus (83) and subsequent studies intoARFs function, provide a possible explanation for the hypersensitivityto overall killing observed in these cell lines. ARF up-regulates p53 inresponse to oncogene activation, includingE1A (84) andmyc (85),resulting in activation ofp53 target genes and cell cycle arrest orapoptosis (86). However, ARF is not required for p53 induction afterDNA damage (87). In the previously describedp53-wild-type MEFsexpressingE1A,there are thus two separate pathways that activate p53and p53-dependent apoptosis. The fact that both of these pathways arefunctional in these minimally transformed cells provides a synergythat can account for the dramatically increased propensity of thesecells to undergo apoptosis after genotoxic or nongenotoxic stress (84).In these hypersensitive cells, modulation of overall cell killing occurswhen one of the pathways is disrupted,e.g.,loss ofp53. It is importantto realize that the loss ofp53 in the transformed MEFs results not somuch in resistance to treatment as much as it does in an eliminationof the unusually hypersensitive response (in this case, due to apopto-sis) that is found after expression ofE1A. Thep53 null transformedMEFs revert back to an overall sensitivity that is closer to theuntransformed parentalp53wild-type cells. In other words, in normalMEFs, p53 modulates the hypersensitive phenotype induced by on-cogenic activation. Interestingly, this synergy also appears to be lostin E1A transformed cells that have lostARFbut retain wild-typep53(84).

An illustration of the role that apoptosis can play in minimallytransformed cells was demonstrated recently using Rat1 cells engi-neered to conditionally expressmyc (72). In this study, induced5 Unpublished data.

Fig. 4. p53 status does not affect the sensitivity ofhuman tumor cells to killing by ionizing radiation. Cellsensitivity (surviving fraction after 2 Gy) was assessed bycolony formation. Results shown inA are from primarytumor cell lines derived from head and neck cancers (46).In these studies, thep53gene was sequenced to determinethose tumors expressing wild-type or mutant p53. Resultsin B show sensitivity of p53-expressingversusp53-non-expressing primary cultures from ovarian cancers (F) andmelanoma (Œ; 45). In these studies, high levels of p53expression were used as a surrogate forp53mutation.l,mean;bars,61 SE for each population.

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expression ofmyc increased thein vitro radiation sensitivity to bothapoptosis and clonogenic survival, and this increased sensitivity wasinhibited by expression ofbcl-2. When grown as tumors in mice,expression ofmycenhanced the sensitivity of these cells to fraction-ated doses of radiation, and this was abrogated by expression ofbcl-2,indicating that apoptosis was directly influencing tumor response toradiation. This experiment thus provides direct evidence that expres-sion of oncogenes in cells originally isolated from normal tissues caninduce an apoptotically sensitive phenotype that is hypersensitive totreatment with anticancer therapies. Becausemychas also been shownto activateARF (85), it is possible that the enhanced sensitivityobserved in these cells and tumors is also due to a synergy betweenoncogene-dependent and DNA damage-dependent pathways to p53induction and p53-dependent apoptosis.

It has been studies such as these using engineered minimallytransformed cells that have, in large part, shaped the current opinionthat apoptosis is also an important determinant of the response ofhuman tumor cells to cancer therapy. As discussed earlier, it has ledto the notion that a tumor cell with mutantp53will be more resistantthan the same cell with wild-typep53, or that increased expression ofbcl-2 in cancer cells will be protective. Acceptance of this hypothesisassumes that cells derived from human tumors will behave similarlyto the virally or oncogenically transformed normal cells describedabove. However, a clear distinction between tumor cells and geneti-

cally engineered cells from normal tissues is the requirement of thetumor cells, during their evolution, to overcome any apoptoticallysensitive state that may have been induced by initial oncogenictransformation. The selection against this apoptotically sensitive stateis driven by the selective forces produced by microenvironmentalstresses such as hypoxia (82), reduced growth factor and nutrientsupply (76), and the requirement for anchorage independent survival(88, 89; Fig. 5).

It is, therefore, likely that the majority of tumors have evolved pastthe point where they may have been apoptotically hypersensitive togenotoxic and nongenotoxic stress. In explanted MEFs expressingviral or cellular derived oncogenes such asE1Aor myc, this selectionoccurs rapidly, and elimination of the sensitive apoptotic state occursby loss of eitherp53 or ARF. In human tumors, this phenotypicevolution can occur with or without alteration of genes such asp53orthose in thebcl-2 family but is associated with a loss of the rapidinduction of apoptosis after genotoxic damage. This selection does notnecessarily eliminate the ability of the cell to carry out apoptosisaltogether; in some cases, the majority of tumor cells may still die bythis process, although usually in a more delayed manner occurringseveral days after treatment and usually after cell division (23, 26, 90).Selection against the apoptotically sensitive phenotype is likely theexplanation for our findings in the present review that apoptosis inhuman tumor cells has minimal impact on the overall cellular sensi-

Fig. 5. Malignant evolution and apoptotic sensitivity. Most differentiated adult cells, as well as normal mouse and human fibroblast cell lines, are resistant to the induction ofapoptosis by genotoxic agents (panel 1). These cells typically undergo permanent arrest or senescence after treatment. Initial oncogenic transformation increases the proliferativepotential and can dramatically sensitize these cells to apoptosis (panel 2). In general, these cells are more sensitive to apoptosis induced by both genotoxic and nongenotoxic stress(e.g.,hypoxia, growth factor withdrawal, or anchorage independence). Cells in this state have also been shown to be hypersensitive to overall cell killing as assessed by clonogenicassay. Examples of cells that can be classified within this apoptotically sensitive state include theE1A-andras-transformed MEFs, the myc-expressing Rat-1 cells, many human stemcells, and hematopoietic tumor cells. Most solid human tumors and tumor-derived cell lines have evolved past this apoptotically hypersensitive state due to the selective pressure arisingfrom various forms of nongenotoxic stress found within the tumor microenvironment (panel 3). In response to genotoxic stress, the predominant mode of cell death for cells in thisstate may or may not be apoptosis. Manipulations in the levels of apoptosis by means of genetic changes has only been shown to affect cells that are apoptotically sensitive (middlepanel). Dramatic changes in the level of apoptosis in cells from human tumors often have no effect on overall survival.

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tivity to present anticancer therapy. Several of these studies showedthat even in cases where the level of apoptosis can be modified inhuman tumor cell lines byp53(60, 62),p21(22, 23), orbcl-2 (20, 21,36), the overall sensitivity to various genotoxic agents was unrelatedto the level of apoptosis. This finding indicates that in these cases,apoptosis after genotoxic treatment occurs only in cells that havealready lost their capacity to continue growth, likely as a result ofunrepaired or misrepaired DNA lesions (Fig. 6).

The studies with oncogenically transformed normal cells, however,do suggest the exciting possibility of improving cancer therapy byexploiting apoptosis. Development of therapeutic agents that are ableto revert human tumors to an apoptotically sensitive phenotype asso-ciated with initial transformation,e.g., by reactivation of pathwayslike the one described above in which ARF participates, would allowuse of current cancer therapies with improved therapeutic potential.The key to the development of such therapies is an increased under-standing of the genetic changes that occur both during initial onco-genic transformation as well as during the selection process thatfollows. However, equally important will be the use of assays thatcorrectly identify therapies that affect the overall level of cell kill as

opposed to those that simply measure changes in the kinetics or modeof cell death.

Summary

In this review, we have focused on the specific question of whetherthe sensitivity of cancer cells from nonhematological malignancies toanticancer drugs and radiation is affected by their p53 status and/ortheir ability to undergo apoptosis after exposure. We find that ifclonogenic survival is used as the end point for cell killing, neitherp53 status nor the ability of the cells to undergo apoptosis appears toplay a significant role in the sensitivity of these cells to DNA-damaging agents. This conclusion is contrary to the widely held tenetthat tumor cells with mutations inp53 and/or that are resistant toapoptosis are also resistant to cancer treatment. Two principal factorshave led to this view: (a) Many studies have used short-term assays,rather than clonogenic survival, to assess cell killing. Because apo-ptosis, particularly p53-dependent apoptosis, can occur rapidly afterexposure, short-term assays tend to underestimate overall killing forcells with mutant p53 or that do not undergo apoptosis. (b) To use

Fig. 6. Two models that illustrate the pathways to cell death in apoptotically sensitive and apoptotically insensitive cells (as defined in Fig. 5) are shown. For apoptotically sensitivecells, genotoxic damage can signal an immediate apoptotic response (A). This signal is dependent upon many genes includingp53, p21, Bcl-2,andpRb. Cells that avoid death at thispoint undergo DNA repair, resulting in cells that have clonogenic potential, and those that do not. In those cells destined to die at this point, a secondary apoptotic decision is made.At this point, cells may either undergo apoptosis or die by other means such as reproductive or necrotic cell death. For apoptotically insensitive cells (B), which comprise the majorityof cells from solid tumors, the primary apoptotic decision point is disabled. After repair, cells that are destined to die still undergo a secondary apoptotic decision point, and thus thepredominant mode of cell death may or may not be apoptosis. The genes controlling apoptosis at both decision points are similar, and thus it is possible to modulate the levels ofapoptosis in cells within the apoptotically insensitive state by genetic means (e.g.,introduction ofbcl-2). However, modulation of apoptosis at this secondary point affects only the modeof cell death, not the overall fraction of surviving cells.

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genetically matched cell lines, investigators have often used normalcells (differing in one specific gene such asp53) transformed withdominant oncogenes as models of cancer cells. Such cells are unusu-ally apoptotically sensitive compared with cancer cells, which haveinvariably undergone a selection process resulting in loss of thishypersensitive phenotype. Extrapolation of data from experimentswith oncogenically transformed normal cells to cells from solid hu-man tumors has thus exaggerated the importance of apoptosis as adeterminant of treatment sensitivity.

Our conclusions that apoptosis and the genes affecting apoptosissuch asp53 and members of theBcl-2 family may not contributesignificantly to the sensitivity of cancer cells to anticancer agents doesnot mean that these genes do not affect the prognosis of humantumors. Tumors with mutatedp53 for example can be more anaplas-tic, have a higher rate of proliferation, and have a more aggressivephenotype than similar tumors with wild-typep53, thereby giving riseto a worse prognosis. Our conclusions also do not apply to cancers ofhematological origin for which apoptosis appears to be the dominantform of cell death after exposure to anticancer agents, nor do theyapply to death receptor (e.g.,Fas)-mediated apoptosis. Finally, the factthat apoptosis is a critical determinant of treatment sensitivity inminimally transformed normal cells warrants further research effortsto understand the loss of apoptotic sensitivity that invariably occursduring solid tumor evolution in hopes of providing more effectivecancer therapy.

Acknowledgments

We thank Dr. Amato Giaccia for helpful comments and Mary Kovacs andDiane Rapaccietta for expert technical assistance.

References

1. McGill, G., and Fisher, D. E. Apoptosis in tumorigenesis and cancer therapy.Frontiers Biosci.,2: 353–379, 1997.

2. Reed, J. C., Miyashita, T., Takayama, S., Wang, H. G., Sato, T., Krajewski, S.,Aime-Sempe, C., Bodrug, S., Kitada, S., and Hanada, M. BCL-2 family proteins:regulators of cell death involved in the pathogenesis of cancer and resistance totherapy. J. Cell Biochem.,60: 23–32, 1996.

3. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. p53 isrequired for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.),362:847–849, 1993.

4. Komarova, E. A., Chernov, M. V., Franks, R., Wang, K., Armin, G., Zelnick, C. R.,Chin, D. M., Bacus, S. S., Stark, G. R., and Gudkov, A. V. Transgenic mice withp53-responsive lacZ: p53 activity varies dramatically during normal development anddetermines radiation and drug sensitivityin vivo. EMBO J.,16: 1391–1400, 1997.

5. Rudin, C. M., and Thompson, C. B. Apoptosis and disease: regulation and clinicalrelevance of programmed cell death. Annu. Rev. Med.,48: 267–281, 1997.

6. Elledge, R. M. Assessing p53 status in breast cancer prognosis: where should you putthe thermometer if you think your p53 is sick? J. Natl. Cancer Inst.,88: 141–143,1996.

7. Falette, N., Paperin, M. P., Treilleux, I., Gratadour, A. C., Peloux, N., Mignotte, H.,Tooke, N., Lofman, E., Inganas, M., Bremond, A., Ozturk, M., and Puisieux, A.Prognostic value ofp53 gene mutations in a large series of node-negative breastcancer patients. Cancer Res.,58: 1451–1455, 1998.

8. Pai, H. H., Rochon, L., Clark, B., Black, M., and Shenouda, G. Overexpression of p53protein does not predict local-regional control or survival in patients with early-stagesquamous cell carcinoma of the glottic larynx treated with radiotherapy. Int. J. Radiat.Oncol. Biol. Phys.,41: 37–42, 1998.

9. Wiggenraad, R., Tamminga, R., Blok, P., Rouse, R., and Hermans, J. The prognosticsignificance of p53 expression for survival and local control in rectal carcinomatreated with surgery and postoperative radiotherapy. Int. J. Radiat. Oncol. Biol. Phys.,41: 29–35, 1998.

10. Cote, R. J., Esrig, D., Groshen, S., Jones, P. A., and Skinner, D. G. p53 and treatmentof bladder cancer. Nature (Lond.),385: 123–125, 1997.

11. Tada, M., Matsumoto, R., Iggo, R. D., Onimaru, R., Shirato, H., Sawamura, Y., andShinohe, Y. Selective sensitivity to radiation of cerebral glioblastomas harboring p53mutations. Cancer Res.,58: 1793–1797, 1998.

12. Bosari, S., and Viale, G. The clinical significance of p53 aberrations in humantumours. Virchows Arch.,427: 229–241, 1995.

13. Weinberg, R. A. How cancer arises. Sci. Am.,275: 62–70, 1996.14. El-Deiry, W. S. The p53 pathway and cancer therapy. Cancer J.,11: 229–236, 1998.15. Lowe, S. W., Bodis, S., McClatchey, A., Remington, L., Ruley, H. E., Fisher, D. E.,

Housman, D. E., and Jacks, T. p53 status and the efficacy of cancer therapyin vivo.Science (Washington DC).,266: 807–810, 1994.

16. Lowe, S. W., Ruley, H. E., Jacks, T., and Housman, D. E. p53-dependent apoptosismodulates the cytotoxicity of anticancer agents. Cell,74: 957–967, 1993.

17. Weinstein, J. N., Myers, T. G., O’Connor, P. M., Friend, S. H., Fornace, A. J., Jr.,Kohn, K. W., Fojo, T., Bates, S. E., Rubinstein, L. V., Anderson, N. L., Buolamwini,J. K., van Osdol, W. W., Monks, A. P., Scudiero, D. A., Sausville, E. A., Zaharevitz,D. W., Bunow, B., Viswanadhan, V. N., Johnson, G. S., Wittes, R. E., and Paull,K. D. An information-intensive approach to the molecular pharmacology of cancer.Science (Washington DC),275: 343–349, 1997.

18. Aldridge, D. R., Arends, M. J., and Radford, I. R. Increasing the susceptibility of therat 208F fibroblast cell line to radiation-induced apoptosis does not alter its clono-genic survival dose-response. Br. J. Cancer,71: 571–577, 1995.

19. Han, J. W., Dionne, C. A., Kedersha, N. L., and Goldmacher, V. S. p53 status affectsthe rate of the onset but not the overall extent of doxorubicin-induced cell death inrat-1 fibroblasts constitutively expressing c-Myc. Cancer Res.,57: 176–182, 1997.

20. Lock, R. B., and Stribinskiene, L. Dual modes of death induced by etoposide inhuman epithelial tumor cells allow Bcl-2 to inhibit apoptosis without affectingclonogenic survival. Cancer Res.,56: 4006–4012, 1996.

21. Kyprianou, N., King, E. D., Bradbury, D., and Rhee, J. G. bcl-2 over-expressiondelays radiation-induced apoptosis without affecting the clonogenic survival of hu-man prostate cancer cells. Int. J. Cancer,70: 341–348, 1997.

22. Waldman, T., Zhang, Y., Dillehay, L., Yu, J., Kinzler, K., Vogelstein, B., andWilliams, J. Cell-cycle arrestversuscell death in cancer therapy. Nat. Med.,3:1034–1036, 1997.

23. Wouters, B. G., Giaccia, A. J., Denko, N. C., and Brown, J. M. Loss of p21Waf1/Cip1sensitizes tumors to radiation by an apoptosis-independent mechanism. Cancer Res.,57: 4703–4706, 1997.

24. Thompson, L. H., and Suit, H. D. Proliferation kinetics of x-irradiated mouse L cellsstudied with time-lapse photography II. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem.Med., 15: 347–362, 1969.

25. Schneiderman, M. H., Hofer, K. G., and Schneiderman, G. S. Anin vitro 125IUdR-release assay for measuring the kinetics of cell death. Int. J. Radiat. Biol.,59:397–408, 1991.

26. Vidair, C. A., Chen, C. H., Ling, C. C., and Dewey, W. C. Apoptosis induced byX-irradiation of rec-myc cells is postmitotic and not predicted by the time afterirradiation or behavior of sister cells. Cancer Res.,56: 4116–4118, 1996.

27. Puck, T. T., and Markus, P. I. Action of X-rays on mammalian cells. J. Exp. Med.,103: 653–666, 1956.

28. Durand, R. E. Cure, regression and cell survival: a comparison of common radiobi-ological endpoints using anin vitro tumour model. Br. J. Radiol.,48: 556–571, 1975.

29. Reinhold, H. S., and De Bree, C. Tumour cure rate and cell survival of a transplant-able rat rhabdomyosarcoma following X-irradiation. Eur. J. Cancer,4: 367–374,1968.

30. Skipper, H. E., Schabel, F. M., and Wilcox, W. S. Experimental evaluation ofpotential anticancer agents. XIII. On the criteria and kinetics associated with “cur-ability” of experimental leukemias. Cancer Chemother. Rep.,35: 1–111, 1964.

31. Begg, A. C. Principles and practice of the tumor growth delay assay.In: R. F.Kallman (ed.), Rodent tumor models in experimental cancer therapy, pp. 114–121.Oxford, England: Pergamon Press, 1987.

32. Wong, C. S., and Hill, R. P. Experimental radiotherapy.In: I. F. Tannock and R. F.Hill (eds.), The Basic Science of Oncology, Ed. 3, pp. 322–349. New York: McGraw-Hill, 1998.

33. Lamb, J. R., and Friend, S. H. Which guesstimate is the best guesstimate? Predictingchemotherapeutic outcomes. Nat. Med.,3: 962–963, 1997.

34. Gura, T. Cancer models: systems for identifying new drugs are often faulty. Science(Washington DC),278: 1041–1042, 1997.

35. Boyer, M. J., and Tannock, I. F. Cellular and molecular basis of chemotherapy.In:I. F. Tannock and R. F. Hill (eds.), The Basic Science of Oncology, Ed. 3, pp.350–369. New York: McGraw-Hill, 1998.

36. Yin, D. X., and Schimke, R. T. BCL-2 expression delays drug-induced apoptosis butdoes not increase clonogenic survival after drug treatment in HeLa cells. Cancer Res.,55: 4922–4928, 1995.

37. Dewey, W. C., Ling, C. C., and Meyn, R. E. Radiation-induced apoptosis: relevanceto radiotherapy. Int. J. Radiat. Oncol. Biol. Phys.,33: 781–796, 1995.

38. Zamble, D. B., Jacks, T., and Lippard, S. J. p53-dependent and -independent re-sponses to cisplatin in mouse testicular teratocarcinoma cells. Proc. Natl. Acad. Sci.USA, 95: 6163–6168, 1998.

39. Potten, C. S. What is an apoptotic index measuring? A commentary. Br. J. Cancer,74:1743–1748, 1996.

40. Gallardo, D., Drazan, K. E., and McBride, W. H. Adenovirus-based transfer ofwild-type p53gene increases ovarian tumor radiosensitivity. Cancer Res.,56: 4891–4893, 1996.

41. Gjerset, R. A., and Sobel, R. E. Treatment resistance, apoptosis, andp53 tumorsuppressor gene therapy.In: J. R. Bertino (ed.), Encyclopedia of Cancer, pp. 1785–1791. San Diego: Academic Press, 1997.

42. Peacock, J., Chung, S., Benchimol, S., and Hill, R. P. Mutant p53 increases radiore-sistance in rat embryo fibroblasts simultaneously transfected with HPV16–E7 and/oractivated H-ras. Oncogene,9: 1527–1536, 1994.

43. McIlwrath, A. J., Vasey, P. A., Ross, G. M., and Brown, R. Cell cycle arrests andradiosensitivity of human tumor cell lines: dependence on wild-type p53 for radio-sensitivity. Cancer Res.,54: 3718–3722, 1994.

44. Siles, E., Villalobos, M., Valenzuela, M. T., Nunez, M. I., Gordon, A., McMillan,T. J., Pedraza, V., and Ruiz de Almodovar, J. M. Relationship between p53 status andradiosensitivity in human tumour cell lines. Br. J. Cancer,73: 581–588, 1996.

45. Zaffaroni, N., Benini, E., Gornati, D., Bearzatto, A., and Silvestrini, R. Lack of acorrelation between p53 protein expression and radiation response in human tumorprimary cultures. Stem Cells,13: 77–85, 1995.

1398

APOPTOSIS, p53, AND TUMOR CELL SENSITIVITY

Research. on March 20, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

46. Brachman, D. G., Beckett, M., Graves, D., Haraf, D., Vokes, E., and Weichselbaum,R. R. p53 mutation does not correlate with radiosensitivity in 24 head and neck cancercell lines. Cancer Res.,53: 3667–3669, 1993.

47. Jung, M., Notario, V., and Dritschilo, A. Mutations in thep53 gene in radiation-sensitive and -resistant human squamous carcinoma cells. Cancer Res.,52: 6390–6393, 1992.

48. Ribeiro, J. C., Barnetson, A. R., Fisher, R. J., Mameghan, H., and Russell, P. J.Relationship between radiation response and p53 status in human bladder cancercells. Int. J. Radiat. Biol.,72: 11–20, 1997.

49. Zolzer, F., Hillebrandt, S., and Streffer, C. Radiation induced G1-block and p53 statusin six human cell lines. Radiother. Oncol.,37: 20–28, 1995.

50. Biard, D. S., Martin, M., Rhun, Y. L., Duthu, A., Lefaix, J. L., May, E., and May, P.Concomitantp53 gene mutation and increased radiosensitivity in rat lung embryoepithelial cells during neoplastic development. Cancer Res.,54: 3361–3364, 1994.

51. Servomaa, K., Kiuru, A., Grenman, R., Pekkola-Heino, K., Pulkkinen, J. O., andRytomaa, T. p53 mutations associated with increased sensitivity to ionizing radiationin human head and neck cancer cell lines. Cell Prolif.,29: 219–230, 1996.

52. Pardo, F. S., Su, M., Borek, C., Preffer, F., Dombkowski, D., Gerweck, L., andSchmidt, E. V. Transfection of rat embryo cells with mutant p53 increases theintrinsic radiation resistance. Radiat. Res.,140: 180–185, 1994.

53. Gupta, N., Vij, R., Haas-Kogan, D. A., Israel, M. A., Deen, D. F., and Morgan, W. F.Cytogenetic damage and the radiation-induced G1-phase checkpoint. Radiat Res.,145: 289–298, 1996.

54. Bristow, R. G., Jang, A., Peacock, J., Chung, S., Benchimol, S., and Hill, R. P. Mutantp53 increases radioresistance in rat embryo fibroblasts simultaneously transfectedwith HPV16–E7 and/or activated H-ras. Oncogene,9: 1527–1536, 1994.

55. Yount, G. L., Haas-Kogan, D. A., Vidair, C. A., Haas, M., Dewey, W. C., and Israel,M. A. Cell cycle synchrony unmasks the influence of p53 function on radiosensitivityof human glioblastoma cells. Cancer Res.,56: 500–506, 1996.

56. Tsang, N. M., Nagasawa, H., Li, C., and Little, J. B. Abrogation of p53 function bytransfection of HPV16E6 gene enhances the resistance of human diploid fibroblaststo ionizing radiation. Oncogene,10: 2403–2408, 1995.

57. DeWeese, T. L., Walsh, J. C., Dillehay, L. E., Kessis, T. D., Hedrick, L., Cho, K. R.,and Nelson, W. G. Human papillomavirus E6 and E7 oncoproteins alter cell cycleprogression but not radiosensitivity of carcinoma cells treated with low-dose-rateradiation. Int. J. Radiat. Oncol. Biol. Phys.,37: 145–154, 1997.

58. Hendry, J. H., Adeeko, A., Potten, C. S., and Morris, I. D. p53 deficiency producesfewer regenerating spermatogenic tubules after irradiation. Int. J. Radiat. Biol.,70:677–682, 1996.

59. Hendry, J. H., Cai, W. B., Roberts, S. A., and Potten, C. S. p53 deficiency sensitizesclonogenic cells to irradiation in the large but not the small intestine. Radiat. Res.,148: 254–259, 1997.

60. Huang, H., Li, C. Y., and Little, J. B. Abrogation of p53 function by transfection ofHPV16 E6 gene does not enhance resistance of human tumour cells to ionizingradiation. Int. J. Radiat. Biol.,70: 151–160, 1996.

61. Slichenmyer, W. J., Nelson, W. G., Slebos, R. J., and Kastan, M. B. Loss of ap53-associated G1 checkpoint does not decrease cell survival following DNA dam-age. Cancer Res.,53: 4164–4168, 1993.

62. Westphal, C. H., Rowan, S., Schmaltz, C., Elson, A., Fisher, D. E., and Leder, P. atmand p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistanceto acute radiation toxicity. Nat. Genet.,16: 397–401, 1997.

63. Zellars, R. C., Naida, J. D., Davis, M. A., and Lawrence, T. S. Effect of p53overexpression on radiation sensitivity of human colon cancer cells. Radiat. Oncol.Investig.,5: 43–49, 1997.

64. Merritt, A. J., Allen, T. D., Potten, C. S., and Hickman, J. A. Apoptosis in small intestinalepithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associatedcell death after gamma- irradiation. Oncogene,14: 2759–2766, 1997.

65. Kawashima, K., Mihara, K., Usuki, H., Shimizu, N., and Namba, M. Transfectedmutantp53 gene increases X-ray-induced cell killing and mutation in human fibro-blasts immortalized with 4-nitroquinoline 1-oxide but does not induce neoplastictransformation of the cells. Int. J. Cancer,61: 76–79, 1995.

66. Pellegata, N. S., Antoniono, R. J., Redpath, J. L., and Stanbridge, E. J. DNA damageand p53-mediated cell cycle arrest: a reevaluation. Proc. Natl. Acad. Sci. USA,93:15209–15214, 1996.

67. Bristow, R. G., Hu, Q., Jang, A., Chung, S., Peacock, J., Benchimol, S., and Hill, R.Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation. Oncogene,16: 1789–1802, 1998.

68. Fan, S., Chang, J. K., Smith, M. L., Duba, D., Fornace, A. J., Jr., and O’Connor, P. M.Cells lacking CIP1/WAF1 genes exhibit preferential sensitivity to cisplatin andnitrogen mustard. Oncogene,14: 2127–2136, 1997.

69. Aldridge, D. R., and Radford, I. R. Explaining differences in sensitivity to killing byionizing radiation between human lymphoid cell lines. Cancer Res.,58: 2817–2824,1998.

70. Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W.Participation of p53 protein in the cellular response to DNA damage. Cancer Res.,51:6304–6311, 1991.

71. Brown, J. M. NCI’s anticancer drug screening program may not be selecting forclinically active compounds. Oncol. Res.,9: 213–215, 1997.

72. Rupnow, B. A., Murtha, A. D., Alarcon, R. M., Giaccia, A. J., and Knox, S. J. Directevidence that apoptosis enhances tumor responses to fractionated radiotherapy. Can-cer Res.,58: 1779–1784, 1998.

73. Ling, C. C., Guo, M., Chen, C. H., and Deloherey, T. Radiation-induced apoptosis:effects of cell age and dose fractionation. Cancer Res.,55: 5207–5212, 1995.

74. Lowe, S. W., Jacks, T., Housman, D. E., and Ruley, H. E. Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells. Proc. Natl. Acad.Sci. USA,91: 2026–2030, 1994.

75. Sanchez-Prieto, R., Quintanilla, M., Cano, A., Leonart, M. L., Martin, P., Anaya, A., andRamon y Cajal, S. Carcinoma cell lines become sensitive to DNA-damaging agents by theexpression of the adenovirusE1Agene. Oncogene,13: 1083–1092, 1996.

76. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M.,Waters, C. M., Penn, L. Z., and Hancock, D. C. Induction of apoptosis in fibroblastsby c-myc protein. Cell,69: 119–128, 1992.

77. Pan, H., and Griep, A. E. Altered cell cycle regulation in the lens of HPV-16 E6 orE7 transgenic mice: implications for tumor suppressor gene function in development.Genes Dev.,8: 1285–1299, 1994.

78. Guo, M., Chen, C., Vidair, C., Marino, S., Dewey, W. C., and Ling, C. C. Charac-terization of radiation-induced apoptosis in rodent cell lines. Radiat. Res.,147:295–303, 1997.

79. Hermeking, H., and Eick, D. Mediation of c-myc-induced apoptosis by p53. Nature(Lond.), 265: 2091–2093, 1994.

80. Bissonnette, R. P., Echeverri, F., Mahboubi, A., and Green, D. R. Apoptotic cell deathinduced by c-myc is inhibited by bcl-2. Nature (Lond.),359: 552–554, 1992.

81. Fanidi, A., Harrington, E. A., and Evan, G. I. Cooperative interaction between c-mycandbcl-2 proto-oncogenes. Nature (Lond.),359: 554–556, 1992.

82. Graeber, T. G., Osmanian, C., Jacks, T., Housman, D. E., Koch, C. J., Lowe, S. W.,and Giaccia, A. J. Hypoxia-mediated selection of cells with diminished apoptoticpotential in solid tumours. Nature (Lond.),379: 88–91, 1996.

83. Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. Alternative reading framesof the INK4a tumor suppressor gene encode two unrelated proteins capable ofinducing cell cycle arrest. Cell,83: 993–1000, 1995.

84. de Stanchina, E., McCurrach, M. E., Zindy, F., Shieh, S. Y., Ferbeyre, G., Samuelson,A. V., Prives, C., Roussel, M. F., Sherr, C. J., and Lowe, S. W. E1A signaling to p53involves the p19(ARF) tumor suppressor. Genes Dev.,12: 2434–2442, 1998.

85. Zindy, F., Eischen, C. M., Randle, D. H., Kamijo, T., Cleveland, J. L., Sherr, C. J.,and Roussel, M. F. Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev.,12: 2424–2433, 1998.

86. Kamijo, T., Weber, J. D., Zambetti, G., Zindy, F., Roussel, M. F., and Sherr, C. J.Functional and physical interactions of the ARF tumor suppressor with p53 andMdm2. Proc. Natl. Acad. Sci. USA,95: 8292–8297, 1998.

87. Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A.,Grosveld, G., and Sherr, C. J. Tumor suppression at the mouseINK4a locus mediatedby the alternative reading frame product p19ARF. Cell,91: 649–659, 1997.

88. McGill, G., Shimamura, A., Bates, R. C., Savage, R. E., and Fisher, D. E. Loss ofmatrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts.J. Cell Biol.,138: 901–911, 1997.

89. Nikiforov, M. A., Hagen, K., Ossovskaya, V. S., Connor, T. M., Lowe, S. W.,Deichman, G. I., and Gudkov, A. V. p53 modulation of anchorage independentgrowth and experimental metastasis. Oncogene,13: 1709–1719, 1996.

90. Waldman, T., Lengauer, C., Kinzler, K. W., and Vogelstein, B. Uncoupling of Sphase and mitosis induced by anticancer agents in cells lacking p21. Nature (Lond.),381: 713–716, 1996.

1399

APOPTOSIS, p53, AND TUMOR CELL SENSITIVITY

Research. on March 20, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

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