ICANCERRESEARCH56. 1834-1841.April 15. 19961

levels of SSBs3 in response to the topoisomerase poison 4'-(9-acridinylamino)-methanesulfon-m-anisidide, when individual lines are analyzed, there is not a direct relationship between the level of damageand cytotoxicity (4).

Recently, the differential sensitivity of cells to chemotherapy hasbeen suggested to be associated with different susceptibilities toundergoing apoptosis (6—10).Sensitivity to DNA-damaging agentsand to irradiation has been demonstrated to depend upon the expression of wtp53 (7, 8, 11, 12). After DNA damage, wild-type p53protein accumulates in the nucleus of the damaged cells by a mechanism that does not require transcription (13, 14). Activated p53transactivates transcription of p53-dependent genes, waf-1, mdm-2,and gadd45 (15—18).Depending upon the cell type, an elevation ofp53 can result in G1 arrest and/or apoptosis (8, 11, 19, 20). These twocellular outcomes are also influenced by growth factors, oncogenes,and activation of cytokine-like signal transduction pathways (21).

Susceptibility to drug-induced apoptosis is also modulated by afamily of proteins that have homology to conserved regions of thebcl-2 gene (22, 23). Apoptosis is promoted by some of the Bcl-2homobogues (Bax, Bak, Bad, and BclX5; Refs. 24—27), whereasother members of the Bcl-2 family (Bcl-2, Bc1XL, and Bag-l) formdimers with the apoptosis-promoters and inhibit their activity,hence suppressing apoptosis (10, 27—30).The ratio of the variousBcl-2 family members has been suggested to predispose a cell toaccelerated or suppressed apoptosis in response to external stimuli(23, 28).

We have used the topoisomerase II poison etoposide to investigatesensitivity of testicular cancer to apoptosis. Etoposide has been usedboth as a single agent and in combination chemotherapy regimes forthe treatment of testicular cancer (31, 32). p53 and Bcl-2 modulatesusceptibility to etoposide-induced apoptosis. Thymocytes from p53-null mice are relatively resistant to etoposide-induced apoptosis, especially at low drug concentrations (1 1). Similarly, mouse bonemarrow or B cells in which Bcl-2 protein is overexpressed showresistance to etoposide (33, 34). Testicular germ cell tumors areunusual because they contain high levels of p53 protein, usuallyindicative of a stabilized mutant form, but have no abnormalities at thegene level (35, 36). Considering the now-established role of wild-typep53 in DNA-damage-induced apoptosis, this could be one of thedeterminants that explain hypersensitivity of testicular tumors tochemotherapy. In contrast, studies have demonstrated that 30—60%ofbladder tumors have mutations in p53 (37, 38). Bcl-2 is not expressedin normal mouse testis, and there are conflicting reports of its presencein the normal bladder (39, 40). As yet, the presence of Bcl-2 has notbeen described in tumors of these tissues. We have investigated theroles of p53 and Bcl-2 as determinants of chemosensitivity in testicular cancers.

3 The abbreviations used are: SSB, single-strand break; FBA, filter binding assay;FIGE, field inversion gel electrophoresis;IC, inhibitory concentration(derived fromclonogenicassays);cdk, cell cycle-dependentkinase.

ABSTRACT

Metastatic testicular cancers are curable, whereas bladder cancers andmost other solid tumors are not. Cell lines derived from human testicular(GH, GCT27, and 833K) and bladder (RT4, RT112, and HT1376) tumorsretain this differential chemosensitivity in vitro. We have investigated thehypothesis that differential sensitivity to chemotherapy is related to dii

ferences in the threshold of susceptibility to undergoing apoptosis. Sensi

tivity to etoposide was not directly related to the frequency of DNA strandbreaks. DNA damage was on average 2-fold greater in the testicular than

the bladder tumor cell lines; in contrast, the testicular tumor lines were15-fold more sensitive to etoposide cytotoxicity than the bladder tumor

lines (IC@ values of 19 ±6 versus 293 ±180 g.tM,respectively). Usingequidamaging (550 md equivalents) etoposide treatments, the percentage

of cells that underwent drug-induced apoptosis was on average higher inthe testicular tumor cell lines than the bladder tumor cell lines. Thetesticular tumor lines have two characteristics that could confer sensitivityto drug-induced apoptosis. First, they have functional p53: the product ofthe p53-dependent gene waf-1 was increased after etoposide treatment.Second, the testicular tumor lines expressed relatively high levels of theapoptosis-promoting protein Bax, but there was no expression of thesuppressor of apoptosis Bcl-2. In contrast, only one of the three bladdercell lines (RT4) had functional p53, and all ofthe bladder lines had readilydetectable levels of Bcl-2 and low levels of Bax. In the testicular cell lines,

increases in p53 and p53-transactivated genes were associated with apop

tosis but not arrest in G1. In contrast, in the bladder cell line (RT4),

increases in p53 and Waf-1 were associated with both arrest in G1 andapoptosis. The differences in the ratio of Bax:Bcl-2 could contribute to thedifferential sensitivity of the two tumor types. However, in contrast to

earlier reports, the ratio of Bax and Bcl-2 was not perturbed by DNAdamage.

INTRODUCTION

Drug resistance is a major problem in tumor chemotherapy, andmetastatic cancer is generally incurable. In contrast, testicular tumorsrarely develop resistance, and approximately 80% of patients withmetastatic disease are cured using combination chemotherapy (1).Thus, testicular tumors may provide clues to the successful treatmentof disseminated cancer. Cell lines derived from testicular germ celltumorsmaintain their sensitivityto chemo- and radiotherapyin vitro(2, 3). They are sensitive to a wide range of chemotherapeutic agents,especially those that damage DNA (2). Studies in which the sensitivehuman germ cell tumors have been compared to relatively resistanttumors derived from transitional carcinoma of the bladder have shownthat drug sensitivity is not directly related to drug target interactions(4, 5). The testicular tumors are 5-fold more sensitive to cis-platincytotoxicity but on average have lower levels of initial damage (5).Similarly, although the testicular tumor lines on average have higher

Received7/26/95;accepted2/I 5/96.The costsof publicationof this article were defrayed in part by the paymentof page

charges.This article mustthereforebe herebymarkedads'ertisementin accordancewith18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by Project Grant SP2234 from the Cancer Research Campaign (toC. M. C.).

2 To whom requests for reprints should be addressed. Fax: 0161-275-5600.

1834

Hypersensitivity of Human Testicular Tumors to Etoposide-induced Apoptosis IsAssociated with Functional p53 and a High Bax:Bcl-2 Ratio1

Christine M. Chresta,2 John R. W. Masters, and John A. Hickman

Cancer Research Campaign Molecular and Cellular Pharmacology Research Group. School of Biological Sciences. The University of Manchester, G.38. Stopford Building,Oxford Road. Manchester. M13 9PT IC. M. C.. .1.A. H.]. and institute of Urology and Nephrology, University College London, 67 Riding House Street. London. WIP 7PNIf. R. W. MI. United Kingdom

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

Table I Origin g celllinesCell

lineBiopsy originPriortreatmentTestis

GHGCT27833KNsGCT―

(Primary)NSGCT (Primary)NSGC'I' (Metastasis)None

NoneChemotherapy6Bladder

RT4RT112HT1376TCC'

(Recurrence)TCC (Primary)TCC (Primary)Radioactive

goldNoneNone

FUNCTIONAL p53 IN CHEMOSENSITIVETUMORS

MATERIALS AND METHODS

Cell Culture and Cytotoxicity Assays. The origins of the cell lines aredetailedin Table 1.

All of the cell lines were grown routinely, under identical conditions, in25-cm2 flasks in RPM! 1640 with 10% (v/v) heat-inactivated FCS and 2 mtiglutamine at 37°Cin a humidified atmosphere of 5% CO2 in air. Each cell linewas used over a maximum of 12 passages to minimize changes that occur

during prolongedculture.All cell lineswereroutinelytestedandfoundto bemycoplasma free.

Drug sensitivity was measured by clonogenic assays. One thousand testicular and 500 bladdertumor cells were plated in 6-well plates(2 cm2)andallowed to adhere overnight. They were then treated for 1 h with eitheretoposideor drug vehicle(DMSO).Thedrugwasthenwashedfrom thecellswith three changes of PBS, and the cells were reincubated in fresh medium for10—14days. Colonies were stainedwith methyleneblue, and coloniesofgreater than 50 cells were counted.

DNA Fragmentation FBA. Apoptosiswas quantifiedusing the FBA (41).PercentageDNA fragmentationassayed using the FBA correspondedwithpercentage of apoptosis determined by counting trypan-blue-negative cells

detachedfrom the monolayer.The detachedcells were demonstratedto beapoptoticby Hoechst33258stainingandby analysisof DNA integrity usingagarose gel electrophoresis (see “Results―).

Conventional Electrophoresis of DNA. After drug treatment,apoptoticcells detached from the monolayer. These cells were collected by centrifugation and combined with the monolayer cells that had been detached bytrypsinisation. Cells (I X 106) were washed in PBS then lysed in 250 p3

sarcosyl lysis solution (0.2% sarcosyl, 2 M NaC1, and 0.04 M EDTA; pH 10)

containing proteinase K at 0.5 mg/ml. The lysate was incubated overnight at45°Cthen diluted with one volume of TE (10 mistTris HC1 pH 7.4; 1 mMEDTA) buffer,andDNA wasprecipitatedby additionof two volumesof 95%ethanol ( —20°C).Precipitates were allowed to form at —20°Cfor 30 mm andthencollectedby centrifugationat 4°C.The pelletswereresuspendedin TEand incubated with 20 p@g/mlRNase for 1 h at 37°Cbefore electrophoresis on1% agarose gels in Tris-borate buffer at 40 V overnight.

Field Inversion Electrophoresis of DNA. Supernatantand monolayercells (4 X 106) were collected as described for conventional electrophoresis.They were then prepared for FlOE as described (42). Under the conditions

used, DNA fragments of lO—750-kbp are resolved and all DNA fragments

greaterthan750—800kbpthatenterthegelmigrateatthesamerate.Molecularweight standardsof 50—1000kb and0.1—200kb (SigmaChemicalCo.) wereused to determine the size of DNA fragments.

Western Blotting. After drug treatment, supematant and adherent cellswere combined as described for conventional electrophoresis. Cells (3 X 106)were rinsed in ice-cold PBS containingthe following PIs: aprotinin (1%),benzamidine(1 mM), phenylmethylsulfonylfluoride (1 mM), and trypsin/chymotrypsin inhibitor (10 @g/ml).The cells were then resuspended in 300 p3

of PBSwith P1andsonicatedusinga Soniprep150(MSE) for 10s at 12 @m,allowed to cool, and sonicated again for 10 s at 16 @.sm.Protein concentration

wasdeterminedusinga Bio-Radproteinassaykit, and50 @gof proteinwereused per sample. Samples were boiled for 5 mm in SDS sample buffer and thenelectrophoresedon 0.75-mm polyacrylamideslab gels at 40 V overnight.Proteinsweretransferredto Immobilon-Pmembranein 10mMCAPSbufferat70 V for 3 h. Gels were stainedwith Coomassieblue to confirmeven loading.

Etoposide (pM)

TESTIS

:@ —&.—

(.3 —0@---‘@ —0—-—833K

@- —h--— RT4

:@ —.—.-RT112

@, —U—-—HT137600U

BLADDER

Fig. I . Survival curves for testicular and bladder tumor cell lines exposed to etoposidefor 1h.Meanvaluesof 4 independentexperimentsarepresented.Variationin thedatawasless than 10% of the mean values. The sensitivities of the two cell types were significantlydifferent as determined by the two sample t tests (0.05 > P > 0.02).

Antibodies. The antibodies used for this study were p53, Ab-6 (DOl),from Oncogene science (Cambridge, MA); Waf-l, EA1O Ab, a kind gift from

Dr. W. S.El-Deity (JohnsHopkins,Baltimore,MD); Mdm2,Ab 2A10,a kindgift from Prof. A. Levine (Princeton, NJ); Bax, N-20, from Santa Cruz Biotech(SantaCruz,CA); andBcl-2, Ab-124,from Dako(High Wycombe,England).Goat antirabbitor mousehorseradishperoxidaseconjugatedsecondaryantibodies (Promega, Madison, WI) were used as described by the manufacturers,andbandswerevisualizedusingenhancedchemiluminescencereagentsfromAmersham (Amersham, England).

Alkaline Elution. DNA SSBs were measuredby alkalineelution(pH 12.1)as described by Kohn et a!. (43). Cells in early logarithmic phase growth

(1—2 X l05/ml) were labeled for 30 h with 0.015 ,tCi [‘4C]thymidine/ml

(specific activity, 56mCi/mmol; Amersham, England), washed, and reincu

bated in fresh medium for 2 h. The cells were then exposed to etoposide for 1h. After drugtreatment,cellswererinsedwith ice-coldPBSandthenscrapedinto versene at 0°Cto minimize the possibility of reversal of damage. Cells

were collected by centrifugationand resuspendedin ice-cold PBS. Cells(8 X l0@) were loaded onto each filter and rinsed with 10 ml ice-cold PBS

before lysis. For measurement of DNA, SSBs cells were lysed using a sarcosyl

lysis solution(0.2%sodiumdodecylsarcosine,2 MNaC1,and0.04MEDTA;pH 10)onto2-@tmpolycarbonatefilters(Millipore).DNA waselutedfor 15hat0.04ml/min with tetraethylammoniumhydroxide(pH 12.1)containing0.1%SDS.DNA remainingon the filter and in the filter funnelswas releasedasdescribed (43). The frequency of SSBs induced by etoposide was converted to

rad equivalentsusinga calibrationgraphderivedfrom the numberof SSBsproducedby a known X-raydose.

Flow Cytometry. Cells were treated with an IC50 concentration of etopo

side for 2 h; they were then washedand reincubatedin the presenceofnocodazole (0.4 @sg/ml)for 22 h. Two controls were used, nocodazole aloneand drug vehicle (DMSO). At 24 h, cells were detached from the plastic bytrypsinisation then washed in saline buffer. Cells (1 X l06/per sample) were

fixed in 70% ethanol and storedat 4°Cuntil FACS analysis.Cells wererehydratedin bufferedsaline,treatedwithRNase(500 units/ml)at 37°Cfor 15mm, and finally stainedwith propidium iodide (50 @Wml)for 20 mm. Cellcycleanalysiswasperformedusinga BectonDickinsonfluorescentactivatedcell analyzer,anddatawasanalyzedusingcell lysis software.

RESULTS

Differential Sensitivity to Etoposide

Clonogenic assayswere performed to establish the sensitivity of thecell lines to a 1-h exposure to etoposide. Fig. 1 shows that thetesticular tumor cell lines were significantly more sensitive to etoposide than the bladder cell lines, with no overlap in the IC@s betweenthe two cancer cell types. On average, loss of clonogenicity was16-fold greater in the testicular than in the bladder cell lines, with a

a NSGCT, nonseminomatous germ cell tumor.

b 833K was derived from a patient that had received combination chemotherapyconsisting of actinomycin D, methotrexate, and cyclophosphamide approximately 2months before removal of the tumor sample.

C TCC, transitional-cell carcinoma.

1835

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

DNA damage was determined after a 1 h treatment with etoposide.Colonies werecounted to assessloss of clonogenicity 12—14days after a 1-h pulse treatment withetoposide.Apoptosiswasmeasuredfollowing 4 h continuoustreatment.Testicular

Bladder Difference PvalueDNA

damage 532 ±28.2 270 ±54.4 1.97-fold 0.002 > P > 0.001(RE)

Lossof clonogenicity87.22±6.31 5.43 ±7.68 16-fold P < 0.001(%)

Apoptosis(%) 39.78±6.52 8.6 ±2.25 4.6-fold 0.002> P > 0.001

FUNCI1ONAL p53 IN CHEMOSENSITIVE TUMORS

2—3-foldvariation within each cell type (Table 2). The bladder cellline HT1376 was exceptionallyresistant,showing40% viability aftertreatment with 360 p@ etoposide.

Comparison of Cytotoxicity Produced by EqUidaInaging Dosesof Etoposide

Etoposide-induced DNA damage was measuredby alkaline elutionin the two tumor cell types. DNA damage was higher in the testiculartumors; on average, the frequency of SSBs induced by 2 @tMetoposidewas 3.8-fold greater in the testicular cell lines than in the bladder celllines (Fig. 24). When DNA damage was correlated with cytotoxicity

A B

Table2 Differential sensitivityof testicularand bladdertumor cell lines to 20 puetoposide-induced DNA damage, cytotoxicity, and apoptosis

@LUpU5lUe .+.+.+ .+.• .+ .5., -+ -@@

(1C90) OH0C127$33KR14 R1112HTI3T$ OH OCT27$3315RT4R1112HTI37$TESTIS BLADDER TESTiS BLADDER

Fig. 3. DNA fragmentationanalysisof testicularand bladdercell lines after 4 htreatmentwith drugvehicle(—;DMSO) or etoposide(+; IC@).A, conventionalagarosegelanalysis;B, fieldinversiongelanalysis.

(loss of clonogenicity), the testicular cell lines showed a significantlygreater loss of viability for a given frequency of DNA damage (Fig.2B). This suggests that chemosensitivity is related to post-DNAdamage events. Therefore, we determined whether the susceptibilityto undergoing apoptosis was different between the two cell types.

Induction of Apoptosis with Etoposide

Apoptosis after 4 h Treatment with Etoposide (IC@). Apoptosisis characterized by cell shrinkage, detachment from monolayers, chromatin condensation, and the nonrandom fragmentation of DNA. Todetermine whether apoptosis could be detected in both cell types, theywere treated for 4 h with a concentration of etoposide that reducedclonogenicity by 90% (IC@). After a 4-h drug treatment, a significantpercentage of the cells detached from the monolayer. Detached andmonolayer cells were analyzed by staining with Hoechst 33258 forcondensedchromatin, a characteristic morphological feature of apoptosis, and by trypan blue uptake for necrosis. Trypan blue uptake wasonly evident in HT1376, the exceptionally resistant bladder tumor cellline; in this cell line, only 11% of cells detached, and 63% of thesewere trypan blue positive. In the remaining two bladder cell lines,RT4 and RT112, and in the testicular cell lines, detached cells cxcluded trypan blue and had condensed chromatin. Analysis of DNAintegrity by conventionalelectrophoresisandFlOE revealednonrandom cleavageof DNA in the cell lines that exhibited morphologicalfeatures of apoptosis. Cleavage to 50-kb fragments and intemucleosomal cleavage were detected in all of the testicular lines and in thebladder cell lines RT4 and RT112 (Fig. 3). There was no evidence ofDNA cleavage in HT1376. The >750-kb band of DNA on the FlOEgels represents unresolved fragments and is a product of the etoposide-induced DNA damage (42). The 750 Kb band was present in allcell types, confirming that HT1376 received the etoposide damage butdid not couple it to apoptosis.

Comparison of Susceptibility to Undergoing Apoptosis UsingEquimolar and Equidamaglng Concentrafloos of Etoposide. Apoptosis was quantified for a range of etoposide concentrations using theFBA (41). As shown in Fig. 4, the testicular cell lines are more sensitivethan the bladder cell lines to etoposide-inducedapoptosisat all concentrations studied.However, there was variation in susceptibility to undergoing apoptosiswithin each tumor cell type. The testicular lines 833Kand GH were more sensitive than OCT27; the percentage of DNAfragmentation of these cell lines after a 4-h treatment with 20@

ATESTIS

-@

g —0—GCT27@# —0— 833K

@) —*— RT4

.g —.—-@ —.—

BLADDER

B

*

:@ .@ foNowtng 550RE $58..

g‘4-

I

Fig. 2. Comparisonof etoposide-inducedDNA damageand lossof clonogenicityintesticularand bladdercell lines. A, frequencyof DNA SSBs(tad equivalents)weredeterminedby alkalineelution after 1 h treatmentwith a rangeof etoposideconcentrations. Testicularcell lines@ 0, and 0) and bladder(A, @,and @)cell lines. Datapoints, mean of at least two independent experiments. B, comparison of loss of clonogenicity in testicularandbladdertumorcell linesin responseto DNA damage(550RE).Thesensitivity of the two cell types were statistically significantly different as determined bythe two sample t tests (0.02 > P > 0.01).

1836

Etoposide (pM)

,@.(, r4J rv@ I-

I-@@U@ I—w

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

= F—..@ ‘@-CsJ (0(.9 C―J rn I- ‘- F-..

I— Ci@@ ,- mI.) m 1-(9@ 1-

FUNCFIONAL p53 IN CHEMOSENSITIVETUMORS

most etoposide-resistant bladder cell line, had high basal levels of p53protein that were unchanged after drug treatment. High basal p53protein levels in bladder cancer usually indicate the presence of a p53form stabilized by mutation (44). This is consistent with the absenceof apoptosis and the high level of drug resistance seen in this cell line.

Etoposide-induced Expression of Waf-1 Protein

To determine whether p53 was able to transactivate transcription,levels of the cdk inhibitor Waf-l protein were determined. Waf-l haspreviously been demonstrated to show a p53-dependent increase inresponse to DNA damage (15). Waf-l levels were determined byimmunoblotting of whole cell lysates after treatment with IC10 (90%of cells survive) or IC@ (10% of cells survive) concentrations ofetoposide for 4 h (Fig. 6A). Levels of p53 were also determined forcomparison. Waf-l was found to increase to low levels in the testicular lines OCT27 < GH < 833K and to high levels in RT4, the mostsensitive bladder cell line. A study of the kinetics of p53 and Waf- Iexpression was performed using 833K (testicular) and RT4 (bladder),to determine when p53 and Waf-l levels were maximally induced(Fig. 6B). pS3 protein was maximal after a 2 h treatment, and Waf-llevels were maximal by 4 h. Thus, the absenceof detectable increasesof Waf- 1 in RT1 I2 and HTI 376 (Fig. 6A) is unlikely to be a result ofan inappropriate sampling time. Treatment with low concentrations ofetoposide also resulted in increases in the p53 transactivated geneMdm-2 in the testicular lines and RT4 (data not shown).

Arrest in G1 after Etoposide Treatment

Waf-l is a potent inhibitor of cyclin-dependent kinases. The levelsof this protein increase after DNA damage and have been associatedwith both apoptosis and arrest in G1 (see “Discussion―;Refs. 15, 21,45). The testicular tumor cell lines and the bladder tumor cell line RT4have increased levels of Waf- I protein after etoposide treatment (Fig.6A). However, a G@checkpoint was only observed in the bladder cellline RT4 (Fig. 7). The bladder tumor cell lines that did not haveincreasedWaf-l after etoposidetreatment,RT1 12 and HT1376, didnot undergo a G1 checkpoint (Fig. 7).

Expression of Bax and Bcl-2: Basal Levels and Effect of DNADamage

p53 has been reported to differentially regulate the expression oftwo of the Bcl-2 family members; it promotes the transactivation ofbax, an accelerator of apoptosis, and has been reported to repress theexpression of bcl-2, a suppressor of apoptosis (46, 47). We thereforedetermined whether Bax and Bcl-2 were regulated in the testicular andbladder cell lines after etoposide treatment. Two concentrations ofetoposide were used, IC10and IC@, and sampleswere taken at 4 h, thetime at which Waf-1 levels were maximal (seeFig. 6B). Surprisingly,

65kDa56kDa

38kDa

- + - +- + - + - + - + Etoposide

GH GCT27 833K RT4 RTII2 HT1376 (IC90)

TESTIS BLADDERFig. 5. Etoposide-inducedmodulationof p53 protein levels in testicularand bladder

cell lines.Thecell lineswereincubatedfor 4 h in thepresenceof etoposide(+ ; IC@)ordrug vehicle (—; DMSO). Accumulationof p53 was detectedby immunoblottingasdescribed in “Materialsand Methods.―

80@

60

40@

20@

0

A

us

I

B

us

I

TESTIS

—&.— GH

—0-— GCT27

—a- 833K—&.-— RT4

.—...— RT112

—U-— HT1376

BLADDER

Etoposide (PM)

Fig. 4. Comparisonof etoposide-inducedapoptosisin testicularandbladdercell lines.Apoptosis was quantified by the DNA fragmentation FBA after 4 h treatment withetoposide.A, percentageapoptosisproducedby a rangeof etoposideconcentrationsintesticular (@,0, and 0) and bladder (A, S. and U) cell lines. Data points, mean of at leasttwo independentexperiments.B, comparisonof percentageapoptoticcells at 4 h aftertreatmentwith etoposideconcentrationsresulting in 5SORESSBs.Two samplet testsshowed that the testicular lines were statistically more sensitive to induction of apoptosisthan the bladder cell lines RTI 12 and HT1376 (0.02 > P > 0.01). However, if RT4,which has functional p53, is also included, the difference is less significant(0.1 > P > 0.05).

@.- — —

1837

N %Apoptosis550 RE SSB's

etoposide was 46, 40, and 32%, respectively (Fig. 4). The bladder celllines showed much greater variation in susceptibility to undergoingapoptosis. RT4 was the most sensitive cell line: 34% of DNA wasfragmented after a 4-h treatment with 80 ,.LMetoposide. In contrast,RT112 and HT1376 were relatively resistant to etoposide-induced apoptosis: 4 h treaftnent with 80 pisi etoposideresulted in 19 and 7% DNAfragmentation, respectively. Correlation of the frequency of DNA strandbreaks (rad equivalents) with percentage apoptosis showed that the testicular lines were significantly more sensitiveto apoptosisin responsetoa given level of DNA damage (550RE) than the bladder cell lines RT1 12and HT1376 (Fig. 4 and Table 2).

Expression of p53 in Testicular and Bladder Cell Lines

The absence of mutations in the p53 gene of testicular germ celltumors (35, 36) suggests a potential mechanism for hypersensitivity todrug-induced apoptosis in which wild-type p53 would promote apoptosis. p53 protein levels, in the presence and absence of etoposide,were measured by immunoblotting of whole cell extracts (see Fig. 5).All of the testicular tumor cell lines had, as expected, high basal levelsof p53. However, there was a significant increase of p53 content of3—6fold after etoposide treatment (4 h after an IC@). The bladdertumor cell lines showed variability of p53 expression; RT4 and RT1 12had barely detectable basal levels of p53, but showed the significant12- and 3.5-fold increases after etoposide treatment. HT1376, the

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

FUNCI1ONAL p53 IN CHEMOSENSITIVETUMORS

testicular cancer. The response of solid tumors to chemotherapy isinfluenced by several factors, including tumor size and vascularization. However, cell lines derived from testicular and bladdertumors, when cultured in vitro, retain their relative chemosensitivities and reflect the sensitivities observed in the clinic. This suggests that differential drug sensitivity is an inherent property of thetesticular and bladder tumor cell types (2, 48, 49). We haveinvestigated the threshold of susceptibility to undergoing apoptosisin testicular and bladder tumor cell lines and have determinedwhether this is related to p53 function.

The frequency of DNA damage produced by etoposide was determined and compared to the percentage apoptosis produced by equivalent etoposide treatments. As can be seen in Figs. 2B and 4B, for a

060- • Nocodazole & EtopOsIde

C,!Cl)@.1

-jwC.)

@ 20

orK@r?jj[?

C.)@ I-C,

Fig. 7. Etoposide-inducedG1arrest.Cells weretreatedwith an lC@concentrationofetoposidefor I h; they werethenwashedandreincubatedin thepresenceof nocodazole(0.4 @g/ml)for 22 h. Control cells receivedeither nocodazolealoneor drug vehiclealone(DMSO). Addition of nocodazole prevented cells that were released from an etoposideinduced02 WT@5tfrom moving through the cycle synchronouslyand appearingas anapparent G1 checkpoint. The percentageof cells in G@in the presenceof nocodazole aloneis presented relative to the percentage in G1 after etoposide (1C@) and nocodazole. Cellcycle analysiswas performedusinga BectonDickinson fluorescentactivatedcell analyzer, anddatawereanalyzedusingcell lysis software.

p53 @—@ ———@ —

TESTISBax@ -

C IC IC C IC IC C IC IC Etoposide1090 1090 1090GH GCT27 833K

p53 -@ —@@ @—— —

‘@ ‘ BLADDER

@@ ‘@

Bax@ —@. - @—

C IC IC C IC IC C IC IC Etoposide1090 1090 1090

RT4 RTII2 HT1376Fig. 8. Bax protein is not induced after etoposide treatment. All cell lines were

analyzedfor the inductionof Bax afteretoposidetreatment.Cells weretreatedcontinuously with drug vehicle(C; DMSO), IC1@,or IC@concentrationsof etoposidefor 4 h.

——@@

C IC IC C IC IC1090 1090GH GCT27

—@@@@ —

C IC IC C IC IC C IC IC1090 1090 1090

RT4 RTII2 HT1376

0

Time after etoposide treatment (h)

1838

C IC IC10 90

833K

A

p53 _________

TESTISWaf—1@ —-—‘@

$

Etoposide

p53

BLADDERWaf-1 -@‘@@

@—@@@ â€-̃

Etoposide

B

@‘ —0--— 833K-p53

.@ —0-—— 833K-Waf.1

.@ A RT4-p53

.@ .@ RT4 - Waf-1

Fig. 6. Induction of Waf-l and p53 protein by etoposide. A, comparison of p53 andWaf-l inductionin the testicularandbladdercell linesafter4 h treatmentwith IC30andIC@ dosesof etoposide.Waf-l and p53 were detectedby immunobloningasdescribedin“Materialsand Methods.―B, time course of Waf-l and p53 induction in the testicular cellline 833K and the bladdercell line RT4. Cells were treatedfor I h with an IC@ ofetoposide; they were then washed and reincubated in fresh medium, and samples weretaken at 2, 4, 8, and 18 h. The graph representsband intensitiesderived from theimmunoblot.

Bax protein was not increased in any of the six cell lines under anytreatment conditions (Fig. 8). However, the endogenous levels of Baxwere higher in the testicular tumor lines than in the bladder. To detectBax in the bladder lines, the gel had to be exposed to film for anincreased length of time, resulting in a dark background, as can beseen in Fig. 8. The basal levels of Bcl-2 protein differed dramatically

between testis and bladder. Immunoblotting demonstrated that Bcl-2was present in all of the bladder cell lines but undetectable in thetesticular cell lines (seeFig. 9). There was no detectable Bcl-2 proteinin the testicular lines, even when gels were loaded with high concentrations of protein (150 @.tg)and films overexposed. The levels ofBcl-2 protein in the bladder tumor cell lines were unchanged after a4-h treatment with etoposide (IC@).

DISCUSSION

In the present study we determined whether susceptibility toapoptosis could be the mechanism that confers chemosensitivity to

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

FUNCTIONAL p53 IN CHEMOSENSITIVE TUMORS

Thus, the cell lines that had functional p53 (i.e., all of the testiculartumor cell lines and the bladder cell line RT4) were sensitive toetoposide-induced apoptosis. Mdm-2 expression was also studied as amarker ofp53 function and similar results to those observed for Waf-linduction were obtained in the six cell lines (results not shown).

Increases in p53 after DNA damage have been associated with twocellular outcomes, arrest in G1 and apoptosis (8, 11, 19—21).Thesetwo outcomes are cell type dependent and are probably related toexpression ofthe p53-dependent genewaf-1, a potent inhibitor of cdks(15, 21). Immunoblotting was used to measure Waf-l protein levels inresponse to etoposide-induced DNA damage. In the testicular celllines, there was a transitory increaseof Waf-l to low levels, but no G@checkpoint. However, in one of the bladder cell lines, RT4, there wasa prolonged increase of Waf-l to high levels, and a G1 checkpoint wasobserved (Figs. 6 and 7). The checkpointlapoptosis outcome is affected by the level of the Waf-1 protein (21, 56). Zhang et a!. (56)have shown that greater than one Waf-l molecule is required perternary complex (Waf-l, PCNA, cdk, cyclin) for inhibition of the cdkto occur. Therefore the differences in absolute WaS- 1 protein levelsbetween the testicular and bladder tumor cells could be responsible forthe observed cellular outcomes. The basal levels of Waf-1 are modulated by growth factors and can be regulated by constitutive activation of protein kinases (raf and src) involved in cytokine pathways(21, 57). Further studies are required to determine whether there isdifferential activation of growth-related kinases between the testicularand bladder tumor cell lines. However, src and ras have been described to be activated in bladder carcinomas (58—60) The p53-dependent apoptosis/G1-arrest decision is also modulated by transcription factors involved in cell cycle progression (myc, myb, andE2F). Overexpression of either myc, myb, or E2F results in p53-dependent apoptosis rather than arrest (61—63).N-myc and c-mychave been detected in germ cell tumors (55, 64, 65). Again, furtherstudies are required to determine whether activation of oncogenescontributes to the observed differences in the basal levels of expression of WaI-l and growth arrestlapoptosis in the testicular and bladdertumor cell lines.

The function of p53 in apoptosis is unclear. p53 is both a transactivator and a repressor of transcription, but it is not clear whethereither of theseactivities of p53 are required for apoptosis (45, 66—68).p53 has been described to differentially regulate two of the Bcl-2family membersthat control apoptosis:Bax, an acceleratorof apoptosis, is up-regulated, and Bcl-2, a suppressor of apoptosis, is downregulated (46, 47, 69). This forms an attractive potential mechanismfor p53-induced apoptosis that was supported by studies of Zhan et al.(70), who demonstrated up-regulation of Bax mRNA levels in cellsundergoing apoptosis after genotoxic stress. We determined whetherthe Bax/Bcl-2 ratio was perturbed during etoposide-induced apoptosis. Immunoblotting failed to detect any changes in expression ofeither Bax or Bcl-2 after drug treatment (Figs. 8 and 9). There areseveral potential explanations for the disparity between our results andthose of Miyashita et a!. (46) and Than et a!. (70). First, Bcl-2 and Baxproteins have a long half-life;4 therefore, a modest alteration in proteinsynthesis may not be detected at 4 h. However, there was no increasein Bax RNA at 4 h (data not shown). In Ml murine leukemia cellscontaining a temperature-sensitive p53, incubation at the permissivetemperature for 3 h results in increases in both Bax RNA and protein(47). An alternative explanation is that the increases in Bax expressionare cell type specific; in support of this, Bax protein was not upregulated in Baf-3 cells after activation of p53 (21). Although we didnot observe regulation of these proteins during apoptosis, the endog

l9kDa

+ - Etoposide

(1C90)

lO7kDa

76kDa

52kDa

37kDa

—— . —----- 27kDa

+_ +_ +_ +_

GH GCT27 833K RT4 RTII2 HT1376

TESTIS BLADDERFig. 9. Comparisons of the levels of Bcl-2 in the testicular and bladder cell lines. The

cell lines were incubated for 4 h in the presence of etoposide (+ ; IC@,O)or drug vehicle (—;DMSO).Bcl-2wasdetectedby immunoblottingasdescribedin “Results.―

given frequency of etoposide-induced strand breaks, the testicular celllines die more readily, measuredboth as loss of clonogenicity and aspercentage apoptosis at 4 h (Table 2). This supports the hypothesisthat differential sensitivity is related to differences in the threshold ofsusceptibility to undergoing apoptosis. Within the bladder tumor celllines, there was considerable variation in sensitivity to undergoingapoptosis (Fig. 4A). Two of the lines, RT112 and HT1376, wererelatively resistant, but the third, RT4, showed similar dose-dependentincreases in apoptosis to those seen in the testicular cell lines. Wedetermined whether differential sensitivity to apoptosis was related tospecific differences in p53 function.

p53 plays a critical role in apoptosis after treatment with genotoxicagents (7, 8, 12). Several lines of evidence suggest that p53 functionmay be a determinant of the unusual sensitivity of testicular tumors tochemotherapy. First, testicular tumor cell lines are especially sensitiveto genotoxic drugs; there is little difference in chemosensitivity oftesticular and bladder tumor cell lines to antitumor drugs that inhibitmicrotubule formation (e.g.,. vinblastine and colchicine; Ref. 2).Second, testicular tumors, unlike the majority of solid tumors, do nothave mutations in the p53 gene at the DNA level (35, 36, 50). Finally,there are high levels of p53 protein in testicular germ cell tumors (seeFig. 5; Refs. S1 and 52). The high levels of p53 protein probablyreflect the embryonal origin of these carcinomas. p53 RNA andprotein levels are high in normal embryonic stem cells and undifferentiated embryonic carcinomas (53—55).

p53 was functionally active after addition of etoposide to thetesticular cell lines. Despite the already high levels of p53 present intesticular tumors, DNA damage resulted in a significant increase inp53 protein. This in turn resulted in transactivation of the p53-dependent gene waf-1 (Figs. S and 6). There was considerable vanation in the p53 response of the bladder tumor lines to DNA damage.RT4, the line that was most susceptible to apoptosis, had increasedp53 levels after DNA damage, and this resulted in transactivation ofthe p53-dependent gene waf-1 to high levels. This is in contrast to theapoptosis-resistant bladder cell lines, which did not have functionalp53. Although p53 levels were increased in RTll2 after DNA damage, there was no increase in Waf-l, and HT1376 did not showincreases in p53 or Waf-l (Fig. 6). The p53 protein of RT112 wasshown by immunoblotting to migrate more slowly than the p53 of theother cell lines (Fig. 5). This may be becauseit is a posttranslationallymodified or mutant form. Whichever is the case,the lack of inductionof Waf-l after etoposide treatment suggests that the p53 is inactive. 4 Unpublished data.

1839

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

FUNCTIONAL p53 IN CHEMOSENSITIVETUMORS

enous expression of the two proteins differed significantly betweenthe testicular and bladder tumor cell types. The testicular tumor linesexpressed relatively high levels of the apoptosis-promoting proteinBax, but not the suppressor of apoptosis Bcl-2. In contrast, the bladderlineshad low levelsof Bax andreadily detectablelevelsof Bcl-2. Thedifferences in the ratio of these two proteins could contribute to thedifferential sensitivity of the two tumor types to etoposide (23). Thereare several other Bcl-2 family members that also contribute to drugsensitivity [e.g., Bcl-X (27) and Bak (25)J. Preliminary results have

shown that Bcl-X is expressed in both cell types, but to higher levelsin the bladder. Bak is also expressed in both cell types at the RNA

level.4In summary, the sensitivity of testicular tumors to chemotherapy in

the clinic may, at least in part, be related to the susceptibility of thistumor type to DNA-damage-induced apoptosis. Characterization of

the testicular tumor cell lines at the molecular level revealed threecharacteristics that would contribute to the chemosensitive phenotype.First, the testicular tumors have high levels of p53, presumably in alatent state; p53 further increases in levels after DNA damage andfunctions as a transactivator of transcription. Second, in the testiculartumor cell lines, increases in p53 resulted in apoptosis rather than G1arrest, an outcome presumably dictated by the cell background (as yetnot described for this cell type). Finally, the relative ratio of Bax toBcl-2 would also favor accelerated apoptosis in the testicular celllines, in which, presumably, Bax-Bax homodimers promote celldeath. The absenceof Bcl-2, a pleiotropic suppressor of apoptosis, inthe testicular tumors is a striking difference between the two tumor

types.

REFERENCES

I . Frei, E., III. Curative cancerchemotherapy.Cancer Res., 45: 6532—6537,1985.2. Masters, J. R., Osborne, E. J., walker, M. C., and Parris, C. N. Hypersensitivity of

human testis-tumourcell lines to chemotherapeuticdrugs. Int. J. Cancer, 53: 340—346, 1993.

3. Partis, C. N., Arlett, C. F., Lehmann, A. R., Green, M. H., and Masters, i. R.Differential sensitivitiesto ‘yradiationof humanbladderandtesticulartumourcelllines.mt. j. Radiat.Biol. & Relat.Stud.Phys.Chem.& Med.,53: 599—608,1988.

4. Fry, A. M., Chresta, C. M., Davies, S. M., Walker, M. C., Harriys, A. L., Hartley,J. A., Masters, J. R., and Hickson. I. D. Relationship between topoisomerase IIlevel and chemosensitivity in human tumor cell lines. Cancer Res., 51: 6592—6595, 1991.

5. Bedford, P., Fichtinger-Schepman. A. M. J., Shellard, S. A., Walker, M. C., Masters,J. R. W., and Hill, B. T. Differential repair of platinum-DNA adducts in humanbladderandtesticulartumorcontinuouscell lines.CancerRes.,48: 3019—3024,1988.

6. O'Connor, P. M., Wassermann,K., Sarang,M., Magrath, I., Bohr, V. A., and Kohn,K. W. RelationshipbetweenDNA cross-links,cell cycle, andapoptosisin Burkitt'slymphoma cell lines differing in sensitivity to nitrogen mustard.Cancer Res.. 51:6550—6557,1991.

7. Lowe, S. W., Ruley, H. E., Jacks, T., and Housman, D. E. p53-dependent apoptosismodulatesthe cytotoxicity of anticanceragents.Cell, 74: 957—967,1993.

8. 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.

9. Dive, C., and Hickman, J. A. Drug-target interactions:only the first step in thecommitment to a programmed cell death? Br. J. Cancer, 64: 192—196,1991.

10. Fisher, T. C.. Milner, A. E.. Gregory, C. D., Jackman, A. L., Aherne, 0. W., Hartley,J. A., Dive, C., and Hickman, J. A. Bcl-2 modulation of apoptosis induced by

anticancerdrugs:resistanceto thymidylatestressis independentof classicalresistancepathways.Cancer Rca., 53: 3321—3326,1993.

I I. Clarke, A. R., Purdie, C. A., Harrison. D. J., Moms, R. G., Bird, C. C., Hooper, M. L.,

and Wyllie, A. H. Thymocyte apoptosisinducedby p53-dependentand independentpathways.Nature(Lond.), 362: 849—852,1993.

12. Merritt, A. i., Potten,C. S., Kemp.C. J.. Hickman,J. A., Balmain,A., Lane,D. P.,and Hall, P. A. The role of p53 in spontaneousand radiation-inducedapoptosisin thegastrointestinal tract of normal and p53-deficient mice. Cancer Res., 54: 614—617,1994.

13. Maltzman, W., and Czyzyk, L. UV irradiation stimulates levels of p53 cellular tumorantigenin nontransformedmousecells. Mol. Cell Biol., 4: 1689—1694,1984.

14. Fritsche,M., Haessler,C., and Brandner,0. Inductionof nuclearaccumulationof thetumor-suppressor protein p53 by DNA-damaging agents [published erratum appearsin Oncogene,8: 2605, 19931.Oncogene,8: 307—318,1993.

15. El Deiry, W. S., Harper,1.W., O'Connor,P. M., Velculescu,V. E., Canman,C. E.,Jackman,J., Pietenpol,J. A., Burrell, M., Hill, D. E., Wang, Y., Wiman, K. 0.,Mercer, W. E., Kastan, M. B., Kohn, K. W., Elledge, S. J., Kinzler, K. W.. and

Vogelstein,B. WAFI/CIPI is induced in p53-mediated0, arrestand apoptosis.CancerRes.,54: 1169—1174,1994.

16. Barak,Y., Juven,T., Haffner,R., andOren,M. mdm2expressionis inducedby wildtype p53 activity. EMBO J., 12: 461—468,1993.

17. Perry, M. E., Piette, J., Zawadzki, J. A., Harvey, D., and Levine, A. J. The mdm-2geneis inducedinresponsetoUVlightinap53-dependentmanner.Proc.Nail.Acad.Sci.USA, 90:11623—11627,1993.

18. Than, Q., Bae, I., Kastan,M. B., and Fomace,A. J. The p53-dependent‘y-rayresponseof GADD4S.CancerRes.,54: 2755—2760,1994.

19. Kastan, M. B., Onyekwere, 0., Sidransky, D., Vogelstein, B., and Craig, R. W.Participation of p53 protein in the cellular responseto DNA damage. Cancer Res.,51:6304—6311.1991.

20. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B. Wild-type p53 is acell cyclecheckpointdeterminantfollowing irradiation.Proc.Natl. Acad.Sci.USA,89: 7491—7495,1992.

21. Canman. C. E., Gilmer, T. M., Coutts, S. B., and Kastan, M. B. Growth factormodulationof p53-mediatedgrowth arrest versusapoptosis.Genes& Dev., 9:600—611,1995.

22. Yin, X. M., Oltval, Z. N., and Korsmeyer, S. J. BHI and BH2 domains of Bcl-2 arerequired for inhibition of apoptosis and heterodimerization with Bax. Nature (Lond.),369: 321—323,1994.

23. Korsmeyer, S. J., Shutter, J. R., Veis, D. J., Merry, D. E., and Oltvai, Z. N. Bcl-2/Bax:a rheostatthat regulatesan anti-oxidantpathwayandcell death.Semin. CancerBiol.,4:327—332,1993.

24. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. Bcl-2 heterodimerizes in s'is'owith a conservedhomolog,Bax, that acceleratesprogrammedcell death.Cell, 74:609—619,1993.

25. Farrow, S. N., White, J. H. M., Martinou, I., Raven, T., Pun, K-T., Grinham, C. J.,Martinou, J-C., and Brown, R. Cloning of a Bcl-2 homologue by interaction withadenovirus EIB 19K. Nature (Lond.), 374: 731—733,1995.

26. Yang, E., Zha. .1.,Jockei, J., Boise, L. H., Thompson, C. B., and Korsmeyer, S. J. Bad,a heterodimericpartnerfor Bcl-XL andBcl-2 displacesBax andpromotesdeath.Cell.80: 285—291,1995.

27. Boise, L. H., Gonzalez, 0. M., Postema, C. E., Ding, 1., Lindsten, T., Turka, L. A.,Mao,X., Nunez,0.. andThompson.C. B. bcl-x, a Bcl-2-relatedgenethat functionsas a dominant regulator of apoptotic cell death. Cell, 74: 597—608,1993.

28. Reed,J. C. Bcl-2 and the regulation ofprogrammed cell death. J. Cell Biol., 124: 1—6,1994.

29. Walton, M. I., Whysong, D., O'Connor, P. M., Hockenbery, D., Korsmeyer, S. J., andKohn, K. W. Constitutive expression of human Bcl-2 modulates nitrogen mustard andcamptothecin induced apoptosis. Cancer Res., 53: 1853—1861,1993.

30. Takayama, S., Sato, T., Krajewski, S., Kochel, K., Inc. S., Millan, J. A., and Reed,J. C. Cloning and functional analysis of BAG-l: a novel Bcl-2-binding protein withanti-celldeathactivity. Cell, 80: 279—284,1995.

31. Peckham,M. J., Horwich, A., Blackmore,C., and Hendry, W. F. Etoposideandcisplatin with or without bleomycin as first-line chemotherapyfor patientswithsmall-volumemetastasesof testicularnonseminoma.CancerTreat. Rep.,69: 483—488, 1985.

32. Fitzharris, B. M., Kaye, S. B., Saverymuttu, S., Newlands, E. S., Barrett, A.,Peckham,M. J., and McElwain, T. J. VPI6—213as a single agent in advancedtesticular tumors. Eur. J. Cancer, 16: 1193—1197, 1980.

33. Kamesaki, S., Kamesaki, H., Jorgensen, T. 1., Tanizawa, A., Pommier, Y., andCossman,J. Bcl-2 proteininhibitsetoposide-inducedapoptosisthroughits effectsoneventssubsequentto topoisomeraselI-induced DNA strandbreaksand their repair.CancerRes.,53: 4251—4256,1993.

34. Kondo, S., Yin, D., Takeuchi, J., Morimura, 1., Oda, Y., and Kikuchi, H. Bcl-2 geneenablesrescuefrom in vitro myelosuppression(bone marrow cell death) inducedbychemotherapy.Br. J. Cancer, 70: 421—426,1994.

35. Peng, H. Q., Hogg, D., Malkin, D., Bailey, D., Gallie, B. L., Bulbul, M., Jewett, M.,Buchanan,J., andGoss,P. E. Mutationsof thep53 genedo not occurin testiscancer.Cancer Res., 53: 3574—3578,1993.

36. Heimdal, K., Lothe, R. A., Lystad,S., HoIm, R., Fossa,S. D., and Borresen,A. L Nogermline TPS3 mutations detected in familial and bilateral testicular cancer. GenesChromosomes& Cancer,6: 92—97,1993.

37. Sidransky, D., Von, E. A., Tsai, Y. C., Jones,P., Summerhayes, 1.,Marshall, F., Paul,M., Green,P.,Hamilton,S.R.,Frost,P.,andVogelstein,B. Identificationofp53 genemutations in bladder cancers and urine samples. Science (Washington DC). 252:706—709,1991.

38. Fujimoto, K., Yamada, Y., Okajima, E., Kakizoe, T., Sasaki, H., Sugimura,T., andTerada,M. Frequentassociationof p53 genemutation in invasivebladdercancer.Cancer Res., 52: 1393—1398,1992.

39. Hockenbery, D. M., Zuuer, M., Hickey, W., Nahm, M., and Korsmeyer, S. J. BCL2protein is topographicallyrestrictedin tissuescharacterizedby apoptoticcell death.Proc. Natl. Acad. Sci. USA, 88: 6961—6965,1991.

40. Krajewski, S., Bodnig, S., Gascoyne, R., Berean, K., Krajewska, M., and Reed, J. C.Immunohistochemical analysis of Mcl-l and Bcl-2 proteins in normal and neoplasticlymph nodes.Am. J. Pathol.,145: 515—525,1994.

41. Bertrand,R., Sarang,M., Jenkin, J., Kertigan, D., and Pommier,Y. Differentialinductionof secondaryDNA fragmentationby topoisomeraseII inhibitorsin humantumorcell lineswith amplified c-mycexpression.CancerRes.,51: 6280—6285,1991.

42. Beere, H. M., Chresta, C. M., Alejo-Herberg, A., Skladanowski, A., Dive, C.,Kragh-Larsen, A., and Hickman, J. A. Investigation of the mechanism of higher orderchromatin fragmentation observed in drug-induced apoptosis. Mol. Pitarmacol., 47:986—996,1995.

43. Kohn,K.W.,Erickson,L. C.,Ewig,R.,andFriedman,C.A. Fractionationof DNAfrom mammalian cells by alkaline elution. Biochemistry, 15: 4629—4637,1976.

1840

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

FUNCTIONAL p53 IN C}IEMOSENSITIVETUMORS

44. Kawasaki, T., Tomita, Y., Watanabe, R., Tanikawa, T., Kumanishi, T., and Sato, S.mRNA andproteinexpressionof p53 mutationsin humanbladdercancercell lines.CancerLett., 82: 113—121,1994.

45. Fan,S., El-Deiry, W., Bae,L, Freeman,J., Jondle,D., Bhatia,K., Fornace,A. J.,Magrath,I., Kohn, K. W., andO'Connor,P. M. p53 genemutationsareassociatedwith decreasedsensitivity of human lymphoma cells to DNA damaging agents.CancerRes.,54: 5824—5830,1994.

46. Miyashita, T., Harigai, M., Hanada, M., and Reed, J. C. Identification of a p53-dependentnegativeresponseelementin theBcl-2gene.CancerRes.,54: 3131—3135,1994.

47. Miyashita, T., Krajewski, S., Krajewska, M., Wang, H. G., Lin, H. K., Liebermann,D. A., Hoffman,B., andReed,J.C.Tumorsuppressorp53is a regulatorofBcl-2 andbox gene expression in vitro and in vivo. Oncogene, 9: 1799—1805,1994.

48. Walker, M. C., Parris, C. N., and Masters, J. R. Differential sensitivitiesof humantesticularand bladdertumor cell lines to chemotherapeuticdrugs.J. Natl. CancerInst., 79: 213—216,1987.

49. Sark,M. W. J.,Timmer-Bosscha,H., Meijer.C.,Uges,D. R.A., Sluiter,W. J.,Peters,W. H. M., Mulder, N. H., and deVries, E. G. E. Cellular basis for differentialsensitivity to cis-platin in human gemscell tumour and colon carcinoma cell lines. Br.J. Cancer.71: 684—690,1995.

50. Fleischhacker, M., Strohmeyer, T.. hnai, Y., Slamon, D. J., and Koeffler, H. P.Mutations of the p53 gene are not detectable in human testicular tumors. Mod.Pathol.,7: 435—439.1994.

51. Bartek,J.,Bartkova,J.,Vojtesek,B., Staskova,Z., Lukas,J.,Rejthar,A., Kovarik,J.,Midgley, C. A., Gannon,J. V., and Lane, D. P. Aberrantexpressionof the p53oncoprotein is a common feature of a wide spectrumof human malignancies.Oncogene, 6: 1699—1703,1991.

52.Bartkova,J..Baitek,J.,Lukas,J.,Vojtesek,B.,Staskova,Z.,Rejthar,A.,Kovarik,J.,Midgley, C. A., andLane,D. P. p53 proteinalterationsin humantesticularcancerincludingpre-invasiveintratubulargerm-cellneoplasia.mt. J. Cancer,49: 196—202,1991.

53. Reich, N. C., Oren, M.. andLevine, A. J. Two distinctmechanismsregulatethe levelsof a cellular tumor antigen, p53. Mol. Cell. Biol., 3: 2143—2150,1983.

54. Rogel, A., Popliker, M., Webb, C. G., and Oren, M. p53 cellular tumor antigen.analysis of mRNA levels in normal adult tissues, embiyos. and tumors. Mol. Cell.Biol.,5:2851—2855,1985.

55. Teach,H., Furbass,R., Casper,J., Lyons, J., Bartrans,C. R., Schmoll,H. J., andBronson, D. L Cellular oncogenesin human teratocarcinomacell lines. hit. J.Androl., 13: 377—388,1990.

56. Thang,H., Hannon,0. J., andBeach,D. p2l.containingcycin kinesesexist in bothactiveand inactivestates.Genes& Dev.,8: 1750—1758,1994.

57. Michieli, P., Chedid, M., Un, D., Pierce, J. H., Mercer. W. E., and Givol, D. Inductionof WAF1/C!Pl by a p53-independentpathway.CancerRes.,54: 3391—3395,1994.

58. Fanning, P., Bulovas, K., Saini, K. S., Libertino, J. A.. Joyce, A. D.. andSummerhayes,1.C. Elevatedexpressionof pp6Oc-srcin low-gradehumanbladdercarcinoma.CancerRes.,52: 1457—1462,1992.

59. Burchill, S. A.. Neal, D. E., andLunec,J. Frequencyof H-rasmutationsin humanbladder cancer detected by direct sequencing. Br. J. Urol., 73: 516—521,1994.

60. Levesque, P., Ramchurren, N., Saini, K., Joyce, A., Libertino, J., and Summerhayes,I. C. Screeningof humanbladdertumorsand urine sedimentsfor the presenceofH-mamutations.tnt. J. Cancer,55: 785—790,1993.

61. Wagner,A. J., Kokontis, J. M., and Hay, N. Myc-mediatedapoptosisrequireswild-type p53 in a manner independent of cell cycle arrest and the ability of p53 toinducep2lwafl/cipl. Genes& Dev.,8: 2817—2830,1994.

62. Lin, D., Fiscella,M., O'Connor,P. M., Jackman,J.. Chen,M., Luo, L. L.. Sala,A.,Travail, S., Appella, E., and Mercer, W. E. Constitutive expression of B-myb canbypassp53-inducedWafl/Cipl-mediated G1arrest.Proc.Nail. Acad.Sci. USA, 91:10079—10083,1994.

63. Wu, X., andLevine,A. J. p53andE2F-l cooperateto mediateapoptosis.Proc.Natl.Acad. Sci. USA. 91: 3602-3606, 1994.

64. Misaki, H., Shuin, T., Yao, M., Kubota, Y., and Hosaka, M. Expression ofmyc familyoncogenesin primaiy humantesticularcancer.NipponHinyokikaGakkaiZasshi,80:1509—1513,1989.

65.Watson,J. V., Stewart,J., Evan,0. I., Ritson,A., andSikora,K. Theclinicalsignificanceof flow cytometricc-myc oncoproteinquantitationin testicularcancer.Br.J.Cancer,53:331—337,1986.

66. Caelles, C., Helmberg, A., and Karin, M. p53-dependent apoptosis in the absenceoftranscriptionalactivationof p53targetgenes.Nature(Lond.).370:220—223,1994.

67. Shen, Y.. and Shenk, T. Relief of p53-mediated transcriptionalrepressionby theadenovinis E1B 19 kDa protein or the cellular Bcl-2 protein. Proc. NatI. Acad. Sci.USA,91:8940-8944,1994.

68. Sabbatini,P., Chiou, S. K., Rao, L., and White, E. Modulation of p53-mediatedtranscriptional repression and apoptosis by the adenovirus E1B 19K protein. Mol.CellBiol.,15:1060—1070,1995.

69. Selvakumaran,M., Lin, H. K., Miyashita,T., Wang,H. 0., Krajewski,S.,Reed,J.C.,Hoffman,B.,andUebermann,D.Immediateearlyup-regulationofbaxexpressionbyp53 but not TOF flu: a paradigmfor distinct apoptoticpathways.Oncogene,9:1791—1798,1994.

70. Than,Q., Fan,S., Bae,I., Guillouf, C., Liebermann,D. A., O'Connor, P. M., andFornace,A. J. Inductionof bax by genotoxicstressin humancells correlateswithnormalp53 statusandapoptosis.Oncogene,9: 3743-3751,1994.

1841

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

1996;56:1834-1841. Cancer Res   Christine M. Chresta, John R. W. Masters and John A. Hickman  and a High Bax:Bcl-2 Ratio

p53Etoposide-induced Apoptosis Is Associated with Functional Hypersensitivity of Human Testicular Tumors to

  Updated version

  http://cancerres.aacrjournals.org/content/56/8/1834

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/56/8/1834To request permission to re-use all or part of this article, use this link

Research. on January 24, 2021. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from