[CANCER RESEARCH54, 5054—5058,October 1, 19941

Advances in Brief

The p53-dependent G1 Cell Cycle Checkpoint Pathway and Ataxia-Telangiectasia'

Christine E. Canman, Antonio C. Wolff, Chaw-Yuan Chen, Albert J. Fornace, Jr., and Michael B. Kastan2

The JohnsHopkins OncologyCenter,Baltimore, Maryland 21287(C. E. C., A. C. W., C-Y C., M. B. K.), and Division of CancerTherapeutics,National CancerInstitute,Bethesda,Maryland20892(A. J. F.]

Abstract

The p53 protein is a critical participant in a signal transduction pathway which mediates a G1 cell cycle arrest and apoptotic cell death inmammalian cells after ionizing irradiation. Cells from patients with the

cancer.prone, radiation-sensitive disorder, ataxia-telangiectasia (AT),exhibit suboptimal (delayed and/or defective) induction of p53 protein

after ionizing radiation with some dependence on dose. Other proteinproducts which participate in this signal transduction pathway, includingp21WA@@@l,Gadd4S, and Mdm2, are also suboptimally induced in ATcells after ionizing radiation. Induction ofp53 is also abnormal in AT cellsfollowing treatment with methylmethanesuifonate and bleomycin but ap

pears relatively normal following treatment with UV-C Irradiation or the

topoisomeraseinhibitors,etoposideand camptothecin.Theseresults densonstrate a specific defect in this p53-dependent signal transduction pathway in AT cells. Potential models for this observed specificity of the ATdefect as measured by p53 induction include problems with responses to:(a) single-strand, but not double-strand, DNA breaks; or (b) chemically,

but not enzymatically, generated DNA ends.

Introduction

Transient cell cycle perturbations occur following damage to DNAwith cell cycle arrests noted in the G1, 5, and G2 phases of the cellcycle (1—6).These arrests presumably limit the development of heritable genetic alterations in daughter cells by delaying DNA replication and chromosome segregation until some repair of the DNA hasoccurred. In addition, these arrests may affect the ability of the cell tosurvive the insult to the DNA. A signal transduction pathway, whichmediates the G1 arrest after IR3 and is critically dependent on the p53tumor suppressor gene, has recently been identified (6—11).Ataxiatelangiectasia is a disorder with many phenotypic abnormalities, including hypersensitivity to IR, increased incidence of cancer, progressive cerebellar ataxia with degeneration of Purkinje cells, andabnormalities of many or all of the transient cell cycle arrests whichoccur in normal cells following IR (3, 12—15).We recently demonstrated that cells from AT patients were distinguishable from cellsfrom normal individuals by a failure to induce p53 protein (measuredby immunoblot and immunoprecipitation) 1 h following exposure to 2Gy of IR (7). However, we also showed that loss of the p53-dependent01 arrest does not decrease clonogenic survival after IR (16); thus,

suboptimal p53 induction in AT cells may contribute to geneticinstability but does not appear to account for their increased radiosensitivity (16).

Received 8/18/94; accepted 8/30/94.The costs of publication of this article were defrayed in part by the payment of page

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

1 This work was supported in part by Grant 3187 from the Council for Tobacco

Research, Grants ES05777 and T32CA60441 from the NIH, and the Glaxo ResearchInstitute. M. B. K. is The Steven Birnbaum Scholar of the Leukemia Society of America.C. E. C. andA. C. W. contributedequally to this work.

2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology

Center, 2-109, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-5001.3 The abbreviations used are: IR, ionizing radiation; AT, ataxia-telangiectasia; VP-16,

etoposide; MMS, methylmethanesulfonate; CPT, camptothecin.

A recent publication evaluating p53 protein levels by immunofluorescence reported some increase in p53 immunostaining in AT cells—6hours after irradiation with 5 Gy, in contrast to the rapid increasein normal cells, but stated that this result suggests that induction ofp53 is normal in AT cells (17). Other recent reports have supportedour initial conclusions that AT cells are defective in inducing this p53signal transduction pathway (18—20). Here we present data thatclearly demonstrate suboptimal induction of p53 protein following IRwith evaluations of dose-response and time course of induction.Defects in the induction of the p53-dependent gene products,

@ Gadd4S, and Mdm2, after IR further illustrate thisconcept. Evaluation of the p53 response in AT cells following exposure to other types of DNA-damaging agents also provide furtherpotential insights into the nature of the defect in AT.

Materials and Methods

Cells and DNA Damage. Epstein-Barr virus-immortalized lymphoblastoid

cell lines from normal individuals (2184, 536, and 3657), from AT heterozygotes (736, 3187, 3188, 3334, and 3382), and AT homozygotes (718, 719,1526, 3189, and 3332) were all obtained from the Human Genetic Mutant CellRepository (Camden, NJ). Cell line 718 is not shown in the figures for the sakeof simplicity but yielded similar results to the other AT homozygotes. CPT,VP-16, and MMS were obtainedfrom Sigma ChemicalCo., and bleomycinwas acquired from Mead Johnson. IR and UV irradiation were performed asreported previously (7, 9). For some experiments, time points at 9 and 24 hafter IR were performed in addition to the time points at 1, 3, and 6 h but arenot shown here for the sake of simplicity; they resulted in no additionalconclusions. For the chemical treatments, the cells were harvested after 1, 3,and 6 h of continuous exposure. Untreated controls were included in allexperiments.

Immunoblots. Cells were harvested, counted, and solubilized in Laemmlisample buffer as described previously (7, 9). A modified Lowry assay wasused to quantitate relative protein levels in the samples, and extracts wereloaded so that equivalent cell numbers and/or equivalent protein amounts wereloaded in each comparable set of lanes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and wet electrotransfer, equivalentprotein loadingwas confirmed by staining the nitrocellulose paper with fast green as describedpreviously (7, 9). Immunoblotting was performed as described previously (7,9); for each blot, the nitrocellulose paper was cut so that topoisomerase Iprotein from the same gel could be blotted simultaneously as another controlfor equivalent protein loading. Anti-topoisomerase I antibody was obtainedfrom Topogen (Columbus, Ohio), antibodies to p53 (a mixture of Ab-421 andAb-1801) and mdm2 were obtained from Oncogene Science (Uniondale,NY), and antibody to p2l@'@' was obtained from PharMingen(SanDiego, CA). Antibody to gadd45 was generated by immunizing mice withpurified recombinant human gadd45 protein and generation of hybridomasupernatants.4

Results

Levels of p53 protein in lymphoblastoid cells from normal individuals and AT patients were evaluated by immunoblot at various timepoints after exposure to either 2 or 5 Gy of IR. Equal loading of lanes

4 F. Carrier, M. L Smith, and I. Bae, et a!., Characterization and nuclear localization

of humangadd45,a p53-regulatedprotein,submittedfor publication.

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p53 PAThWAY AND ATAXIA-TELANGIECTASIA

NL AT

218433323189TIME(Hrs)0 13 60 1 360 136

@r -@ —@ @__:@__@:_@-i@ -ølopo I

-w@ -@@@@ @-@ -@@ -@ @.. -@ p53

@ ‘@@ ——@ .øTopoI

-.-.@ @L@@ .@@ p53

536 7190 1 3 6 0 1 36

Fig. 1. Levels of p53 protein in normal (NL) andAT cells at varioustimes afterionizing irradiation.Lymphoblastoid cells from normal individuals(2184 and 536) or individuals with AT (3332,3189, 1526, and 719) were unirradiated(0 h) orirradiated with either 2 or 5 Gy of ‘y-radiationandharvested 1, 3, and 6 h after irradiation. Cellularproteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred tonitrocellulose, and immunoblotted for topoisomerase I, as a control for equivalent loading and forp53 protein.

2Gy

5Gy

2Gy

5Gy

15260136

@-

..@ Topo I

.@ p53

i@Topo I

—— ‘*.@ @.@ .:@@ a@p53

was confirmed by probing for levels of topoisomerase I protein, whichdo not change after DNA damage. Increased levels of p53 proteinwere evident within 1 to 3 h after both doses of irradiation in bothnormal cell lines (Fig. 1). Low levels of p53 induction were apparentin all four AT cell lines, but this induction occurred primarily at thelater time points, and the levels of induction were clearly lower thanthose seen in the normal cells (Fig. 1). At higher doses of IR (10—20Gy), levels of p53 induction in AT cells increased to the point wherethe distinction between the levels of p53 induction in normal and ATcells was less clear (data not shown). Induction of p53 protein inlymphoblastoidcell lines generated from four differentAT heterozygoteswas indistinguishablefrom that seen in normal cells (data not shown).

p53 protein activates the transcription of at least three differentgenes following DNA damage: GADD45, MDM2 and p21 WAF1/CIP1(7, 10, 11, 19, 21, 22). We evaluated relative levels of the proteinproducts of these three genes in normal and AT cells at various timesfollowing exposure to various doses of IR. Levels of all three of theseproteins increase after IR in normal cells, but their induction after IRis noticeably less in the four AT cells lines evaluated (Fig. 2). It isinteresting to note that the basal levels of Gadd4S protein appearselevated in the AT cell lines 1526 and 719. However, no change inthese levels is noted following irradiation.The cause of this increasedbasal level of Gadd4S protein has not been determined. Some induction of all three proteins is seen in at least one of the AT cell lines atthis dose of IR; however, in all cases, the induction is either late orquantitatively less than that seen in the normal cell lines. The levels ofinduction of these three proteins at 5 Gy of IR was similar to thatshown here following 2 Gy but, as seen with p53 induction, doses ashigh as 20 Gy seemed to result in higher levels of induction of theseproteins (data not shown). The suboptimal induction in AT cells of allof these proteins after IR, all of which are at least partially dependenton p53, is consistent with the data presented in Fig. 1 that IR inductionof p53 is suboptimal in AT cells.

Levels of p53 protein in normal and AT cell lines were alsoexamined following exposure to several other types of DNA-damag

ing agents. UV-C irradiation (8 J/m2) induced p53 protein in all fourAT cell lines examined to levels thatwere indistinguishablefrom thelevels of induction in two normal cell lines (Fig. 3A). All four AT celllines similarly induced p53 protein following exposure to the topoisomerase I inhibitor, CPT, to levels which were indistinguishablefrom those induced in normal cells (Fig. 3A). Exposure of AT cells tothe topoisomerase II inhibitor, VP-16, resulted in p53 induction (Fig.3B), but in the experimentshown,it is difficult to concludewhetherthere is a small quantitative difference between induction in normaland AT cells. Treatment of normal cells with the alkylating agentMMS resulted in induction of p53 protein at 3 and 6 h after exposure,with no detectable induction at 1 h (Fig. 3A). This result is consistentwith our previous observation of a lack of p53 induction 1 to 2 h afterMMS exposure in other cell types (9). Some induction of p53 wasnoted in AT cell lines 3189, 1526, and 719 after MMS treatment, butthe increased levels were not evident until 6 h after exposure to theMMS and appeared quantitatively less than the induction seen in thenormal cells. The level of p53 induction after MMS treatment in ATcell line 3332 shown here was not convincingly different than thelevel seen in the normal cells. The bulk of the data shown here,however, suggests a quantitatively defective and/or delayed inductionof p53 protein after MMS treatment in AT cells. Exposure of AT cellsto the radiomimetic agent bleomycin revealed a markedly decreasedlevel of p53 induction relative to normal cells (Fig. 3B).

Discussion

We initially reported defective induction of p53 protein by 2 Gy IRin AT cells by evaluating p53 protein levels by immunoprecipitationand immunoblot 1 h following irradiation (7). This time point waschosen because the induction of p53 protein after IR is very rapid(with measurable increases within 10 mlii after DNA damage),5 and

the presumptive role of the G1 arrest would require a rapid cellular

5 M. H. Lee and M. B. Kastan, unpublished observations.

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ATNL3189

0 136

.@ mdm2

33320 1 36

.@@@

‘ .-.

@.i@gadd45.@-.@

.@:@ :—53615267190

1 3 601 3 60 1 36

Fig. 2. Levels of Mdm2, Gadd4S, and@dpi proteins at various times after ionizing irradi

ation. Lymphoblastoid cells from normal (NL;2184 and 536) or AT (3332, 3189, 1526, and 719)individuals were either unirradiated (0 h) or irradiated with 2 Gy of y-irradiation and harvested 1, 3,and 6 h after irradiation. Cellular extracts wereimmunoblotted for either Mdm2, Gadd45, or

@2lwAFi@I@@1 proteins. Equivalent loading was

documented by fast green staining of the nitrocellulose paper (data not shown) and evaluation oftopoisomerase I protein levels (same cellular cxtracts as shown in Fig. 1). Similar results were seenafter exposure to 5 Gy IR (data not shown).

_ -

response for optimal physiological effect. Lu and Lane (17) disputedthe concept that there is a defect in p53 induction after IR in AT cellsbased on experiments using subjective immunofluorescent microscopic evaluations of p53 protein levels. They noted some increases in

p53 immunofluorescence approximately 6 h after exposure to 5 GyIR, while normal cells exhibited increased immunofluorescence muchmore rapidly. Despite the fact that their assays evaluated a limitednumber of cells by subjective analysis, rather than the millions of cellsused by our more objective assays, and despite the obvious differencesin time courses of p53 induction in normal and AT cells after IR, theyconcluded that AT cells are not substantially defective in p53 induction after IR. Dismissing a delay in p53 induction after IR in AT cellsas an insignificant difference from normal cells ignores the potentialcontributions of failing to rapidly inhibit replication of a damagedDNA template to the development of genetic instability and tumorigenesis. In addition, these authors ignored the difference in doses ofIR used in the two reports.

The time courses and dose-responses presented in this report address some of the apparent differences in the two previous publications (see below) and, along with the evaluation of induction ofp53-dependent gene products, confirm the conclusion that p53 proteinis suboptimally induced by IR in AT cells. This conclusion is alsosupported by other recent reports in the literature: (a) Khanna andLavin (18) similarly reported abnormalities in p53 induction after IRin AT cells, with some variation as a function of dose and time; (b)Price and Park (19) reported abnormal IR induction of p53 DNAbinding activity in electrophoretic mobility shift assays and abnormalinduction of MDM2 mRNA after IR; and (c) Dulic et a!. (20) demonstrated failure of inhibition of cyclin/cdk complexes in AT cells byIR, an activity presumably mediated by p21WAFI/clPlinduction.

Three gene products have been identified whose transcriptionalactivation following IR is dependent on p53: GADD4S, MDM2, and

@ (7, 10, 1 1, 19, 21). It has been reported previously that

GADD4S (23) and MDM2 (19) mRNA levels do not increase in ATcells after IR as much as they do in cells from normal individuals.

Here we extended these observations to evaluations of protein levelsof these gene products in a wider range of AT cell lines and similarlyevaluated levels of p21wAFl/CIP1 after IR in AT cells. Decreasedinduction after IR of all three of these gene products was noted in allof the AT homozygote cell lines tested, providing further evidencethat AT cells do not optimally induce this p53-dependent pathwayafter IR.

The nature of the defect in AT remains to be elucidated, but someinsights could be provided by characterization of the types of DNAdamaging agents which elicit normal and abnormal responses in AT.In addition to IR, we found suboptimal p53 induction followingtreatment with the radiomimetic agent, bleomycin, and the alkylatingagent, MMS. In contrast, p53 induction appeared relatively normalfollowing exposure to UV-C irradiation and treatment with the topoisomerase I inhibitor, CPT, and the topoisomerase II inhibitor, VP-16.Both MMS and UV create base damage, but the former is repaired by“shortpatch―excision repair with probable involvement of apurinicendonucleases, whereas the latter results in more significant alter

ations of DNA structure, beginning with the bulky thymidine dimeradduct and being repaired by “longpatch―excision repair (24).Unfortunately, the intermediates in the repair of these agents are notelucidated well enough to conclude why the AT cells can respondrelatively normally to the UV damage with p53 induction but notrespond normally to the MMS damage. Possible explanations includedifferences in recognition of the two types of base damage or differences in the responses to the DNA structural changes induced by thedifferent repair processes. Interestingly, single-strand DNA breaksappear to be a reaction intermediate during the repair of methylatedbases (25).

We have reported previously that DNA strand breaks appear to bethe signal which induces p53 protein levels following DNA damage(9, 22), althoughwe could not makeany distinctionsbetween the roleof single versus double-strand breaks. Interestingly, we found that thatCPT treatment would induce p53 protein levels only if replicationdependent double-strand breaks were introduced into the DNA (9).

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A.

NL AT2184

TIME(Hrs) 0 1 3 63332 0 1 3 63189 0 1 3 6

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CPT@@@@@ _ —a@@ TopoI.@@ —@ -@@ p53

NL AT

— -..@TopoI- $%@@ ...øp53

CPT

@ @__

53601 3 6

uv

Fig. 3. Levels of p53 protein in normal (NL) andAT cells at various times after treatment with different DNA-damaging agents. Lymphoblastoidcells from normal (2184 and 536) or AT (3332,3189, 1526, and 719) individuals were either untreated or treated with: 8 J/m2 UV irradiation, 25

@g/mlMMS or 1 @LMCPT(A); or 10 g.&g/mlVP-16or 0.05 unit/mI bleomycin (Bleo) (B). Cells wereharvested 1, 3, and 6 h after irradiation or additionof drug and cellular extracts were immunoblottedfor topoisomerase I protein, as a loading control,and p53 protein. Inefficient protein transfer wasnoted in the Topo I lanes in 3332 and 3189 at 6 hof CPT treatment and in the p53 lanes in 1526 and719 at 6 h of Bleo treatment.

15260136

B.

2184TIME(Hrs) o 1 3

BLEO

5366 0 1 3 6

1526 719

0 1 3 6 0 1 3 6

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@@ —@@@ ..@@ p53

VP-16@ @r@@ ,@;;;_, I_,_@@ .. .øTopol___ — —@ @,@ — —@@@@@@ p53

Both VP-16 and CPT induce double-strand breaks in the DNA (26,27), whereas y-irradiation primarily induces single-strand DNAbreaks (25, 28). However, a small ratio of double-strand:single-strandbreaks are induced by y-radiation (28); thus, at higher IR doses,greater absolute numbers of double-strand breaks would be present.Bleomycin also induces single-strand DNA breaks (29, 30), but thenature of the oxygen intermediates makes these breaks cluster so thatapparent double-strand breaks are also generated. Again, the absolutenumbers of double-strand breaks would be dose dependent. Thus, onepossible model is that the AT cells are responding appropriately to thepresence of a critical number of double-strand breaks (in this case, asindicated by increasing p53 protein levels) but are responding poorlyto the presence of single-strand DNA breaks. Increasing doses ofagents, like IR or bleomycin, to increase absolute numbers of double

strand breaks could overcome the apparent differences between normal and AT cells as measured by p53 induction.

Mechanisms other than differences in response to single- versusdouble-strand DNA breaks could also account for this spectrum ofDNA-damaging agents which appear to elicit normal versus abnormalresponses in AT. For example, there may be commonalities in theprocessing of these DNA lesions or in the proteins associated with thespecific repair complexes, which could account for this grouping ofagents which are capable of inducing p53 in AT. The observation thatthe three agents tested which are able to induce p53 in AT cells (UV,CPT, and VP-16) all induce strand breaks enzymatically and generateDNA ends that require no further processing, whereas the agentstested which are poor at inducing p53 in AT cells (IR, bleomycin, andMMS) generate DNA breaks with chemical intermediates which must

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p53 PATHWAYAND ATAXIA-TELANGIECFASIA

be further processed, provides another potential explanation for therelative specificity of the AT defect. Further elucidation of the intermediate structures arising following exposure to these agents couldprovide novel insights into the nature of the defect in AT.

Normal mammalian cells arrest at multiple sites in the cell cyclefollowing IR, including G1, S. and G2 (1—3,6, 14). It is important tonote that AT cells appear to be defective in virtually all of these cellcycle arrests (3, 5, 15, 31, 32). The p53-mediated arrest induced by IRoccurs at a discrete stage of G1 (prior to phosphorylation of theretinoblastoma protein; Refs. 10 and 33) and is distinct from the muchmore transient arrest which occurs in S following IR. The lack of the

latter S-phase arrest was the response originally measured in the initialstudies of “radioresistantDNA synthesis―in AT cells, since the DNAsynthesis was measured almost immediately after irradiation andnormal cells recovered rapidly from this arrest (3). In contrast, thep53-mediated arrest appears to last for a much longer period of timeand is evident as decreased DNA synthesis measured at much latertime points (6—8).Unlike the arrest in G1 after IR, the IR-inducedS-phase arrest appears to be independent of p53 (34). Similarly, thearrest of cells in G2 is not dependent on p53 (6). It is likely that the

failure of AT cells to arrest at all of these critical junctions is due to

a single defect, possibly related to suboptimal responses to a specifictype of DNA break. The suboptimal induction of p53 after IR isprobably only one manifestation of this defect and is not likely to beresponsible for all (if any) of the phenotypic abnormalities seen in AT.

However, it appears to be a useful measure of the response capabilitiesof AT cells. It is already clear, for example, that the p53 inductiondefect is not responsible for the radiosensitivity noted in AT (16). Theobservation that AT cells exposed to IR in G2 fail to arrest at the G2-M

checkpoint, whereas AT cells which are irradiated in S are capable ofarresting when they reach G2-M (32), may also reflect such differ

ences in the nature of the DNA breaks induced by IR in the twodifferent cell cycle stages. Further molecular characterization of all ofthese responses in AT should continue to provide new insights into thedefective mechanisms responsible for the phenotypic abnormalitiesseen in this patient population.

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1994;54:5054-5058. Cancer Res   Christine E. Canman, Antonio C. Wolff, Chaw-Yuan Chen, et al.   Ataxia-Telangiectasia

Cell Cycle Checkpoint Pathway and1The p53-dependent G

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