P53 mutation in acute T cell lymphoblastic leukemia is of somatic origin and is stable during establishment of T cell acute lymphoblastic leukemia cell lines

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P53 mutation in acute T cell lymphoblastic

leukemia is of somatic origin and is stable

during establishment of T cell acute

lymphoblastic leukemia cell lines.

J Yeargin, … , M Bogart, M Haas

J Clin Invest.

1993;

91(5)

:2111-2117.

https://doi.org/10.1172/JCI116435

.

Samples donated by patients with T cell acute lymphoblastic leukemia (T-ALL) were

screened for mutations of the p53 tumor suppressor gene. Peripheral blood cells of T-ALL

relapse patient H.A. were found to possess a heterozygous point mutation at codon 175 of

the p53 gene. To determine whether this was an inherited mutation, a B cell line (HABL)

was established. Leukemic T cell lines (HATL) were concurrently established by growing

peripheral blood leukemic T cells at low oxygen tension in medium supplemented with

IGF-I. Previously we had shown that > 60% of leukemic T cell lines possessed mutations in the

p53 gene (Cheng, J., and M. Hass. 1990. Mol. Cell. Biol. 10:5502), mutations that might

have originated with the donor's leukemic cells, or might have been induced during

establishment of the cell lines. To answer whether establishment of the HATL lines was

associated with the induction of p53 mutations, cDNAs of the HATL and HABL lines were

sequenced. The HATL lines retained the same heterozygous p53 mutation that was present

in the patient's leukemic cells. The HABL line lacked p53 mutations. Immunoprecipitation

with specific anti-p53 antibodies showed that HATL cells produced p53 proteins of mutant

and wild type immunophenotype, while the HABL line synthesized only wild-type p53

protein. The HATL cells had an abnormal karyotype, while the HABL cells possessed a […]

Research Article

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(2)

P53 Mutation

in

Acute

T

Cell Lymphoblastic Leukemia Is of Somatic Origin and Is

Stable during

Establishment

of

T

Cell Acute

Lymphoblastic Leukemia Cell

Lines

JoYeargin,* Jian Cheng,* Alice L.Yu,tRuth Gjerset,* MarkBogart,§andMartin Haas*

*University of California San Diego Cancer Center, Departments of Pathology, Biology, tPediatrics, and §Genetics, University of California, San Diego, La Jolla, California 92093-0063

Abstract

Samples donatedby patients with Tcell acute lymphoblastic leukemia (T-ALL)werescreened for mutationsofthep53

tu-mor suppressor gene.Peripheral bloodcellsofT-ALLrelapse patientH.A.werefoundtopossessaheterozygouspoint

muta-tionat codon 175ofthep53gene. To determine whetherthis

was an inherited mutation, a B cell line(HABL) was

estab-lished. LeukemicTcelllines(HATL)wereconcurrently estab-lishedbygrowing peripheralbloodleukemicTcellsatlow oxy-gentensioninmediumsupplemented withIGF-I.Previouslywe

had shownthat>60% of leukemicTcell linespossessed

muta-tionsinthep53gene(Cheng, J., andM.Haas. 1990. Mol. Cell. Biol. 10:5502), mutations thatmighthaveoriginatedwith the

donor's leukemic cells,ormighthavebeeninducedduring

es-tablishment of the cell lines.Toanswerwhether establishment

ofthe HATL lines was associated with the induction of

p53

mutations, cDNAs ofthe HATL and HABL lines were

se-quenced.The HATL lines retained thesameheterozygousp53 mutation thatwaspresent in the

patient's

leukemiccells.The

HABLline lacked p53 mutations.

Immunoprecipitation

with

specific anti-p53

antibodies showedthat HATL cells

produced

p53 proteins ofmutantand wildtypeimmunophenotype, while

the HABL line

synthesized

only wild-type p53 protein. The HATLcellshadanabnormalkaryotype, while theHABL cells

possessedanormal

diploid

karyotype.Theseexperiments sug-gest that (a) p53 mutation occurred in the leukemic cells of

relapse T-ALL patient HA; (b)the mutationwas ofsomatic

ratherthan hereditary origin; (c)the mutation was leukemia associated;and(d) establishment ofhuman leukemiacelllines needsnot beassociated with in vitro induction ofp53

muta-tions.It may be

significant

that

patient

HA

belonged

to a

cate-gory of relapseT-ALL

patients

in whoma secondremission

could not be induced. (J. Clin. Invest. 1993.

91:2111-2117.)

Keywords:acute

lymphoblastic

leukemia-

p53

andTcellacute

lymphoblastic

leukemia relapse*tumorsuppressor gene *

es-tablishment ofleukemia lines*somatic mutation of

p53

Introduction

P53 belongsto thetumorsuppressorclass ofgenes(1) whose loss-of-function mutations are oncogenic. Inactivation of the

Addresscorrespondence toMartinHaas, Ph.D., Department of

Biol-ogy/UniversityofCalifornia, San Diego, Cancer Center,0063,9500

GilmanDrive,LaJolla, CA 92093-0063.

Receivedforpublication 29 April 1992 and in revisedform 15 De-cember 1992.

p53genebypoint mutation,

deletion,

orrearrangementhave been found inawiderange

of

humantumors.Inactivation

of

thep53genehas been demonstrated in human carcinomas

of

the colorectum (2), lung (3, 4), liver (5, 6), bladder (7), and

ovary(8), in chronic myelogenous leukemias (9, 10),in

osteo-genicsarcomas(11, 12), and inTcellacutelymphoblastic leu-kemias

(T-ALL)'

(13, 14). Since in carcinomas of the

colorec-tumchromosome 17pdeletionsareassociated with the transi-tion from the benign adenomatous to the

malignant

carcinomatousstate,ithasbeensuggested that p53

mutation/

deletion isalateeventin the development ofcancer(15). How-ever,

mutations

inp53 have recently been found in

adenoma-tous polyps

of patients

with familial

polyposis

coli (16),

sug-gesting that p53 mutationmay alsooccurasanearlyeventin

carcinogenesis.

Arolefor p53 intheinductionandprogression ofhuman

cancerhasbeen suggestedby thestatusof p53 in individuals withtheLi-Fraumenisyndrome,inwhominherited (germline)

heterozygous mutations in p53 areassociated with astriking predisposition to anumber ofcancers(17, 18). However, in familial leukemia pedigrees

hereditary

p53 mutationshavenot

been found(19).

Inrelapse leukemicTcelllines, both alleles have frequently been foundtobe

independently

mutated, rather thanoneallele

being

mutated, the other deleted (15, 13).Aswehaveproposed

previously (13),

thehigh frequency of mutation of all alleles

of

thep53geneinleukemicTcell linesmaybe duetomutations

that have occurred(a) in vitro,

(b)

invivo, or(c) bothinvivo and in vitro. In previous work (13) we did not determine

whether themutations foundin T leukemiacelllineshad

re-sultedfrom establishment of the lines in culture, ashas been showntooccurduring the establishment ofsome ratembryo fibroblast cell lines (20). Alternatively, the high frequency of mutations in

p53

inTleukemia celllines may be due to the

selective establishment

of

lines fromT-ALLcells that already

possess

p53

mutations in vivo. Presumably, p53 mutationsare presentina

minority

of leukemiacases(21, 14, 19), but appear

inalarge fraction ofTleukemiacelllinesdue tothe advantage

theseleukemiashave

during

in

vitro

establishment.Inthis

sce-nario

themutationsobservedinTleukemia lines would have

originated

in vivo. Athird possibility is thatin T-ALL, p53

mutation isnot generally found in

"diagnosis"

T-ALL cases

(21), but is associated withthe relapse phase, as has been shown

foronecase(14).

Toexaminewhetherp53mutationsplay a role in the

mor-bidity ofT-ALL,weexploredtwomutation"hotspots" in the

p53

gene

(15)

by PCR amplification of genomic DNA and

restrictionenzymedigestion.One patient (H.A.) was shown to possess a mutationat codon 175, and thiscase was studied

1.Abbreviation usedinthis paper: T-ALL,Tcellacutelymphoblastic

leukemia.

J.Clin. Invest.

©TheAmerican Society for Clinical Investigation,Inc.

0021-9738/93/05/2111/07 $2.00

(3)

further. Our data show that this p53 gene mutation waspresent

in peripheral blood nucleated(leukemia)cells of patient H.A., thatthe mutation was retained by long-term T-ALL cell lines

grown continuously for>2 yrfrom H.A.'s peripheral blood

nucleated cells, and that the mutation was somatically

ac-quired. Our experiments also show that additional mutations in the p53 gene were not induced by establishment ofthe leuke-mia cells in vitro.

Methods

PrimaryT-ALLsamples. Bone marrow, or Ficoll-Hypaque-separated

peripheralblood nucleated cells, werefrozenlive inmedium contain-ing 10%DMSO, at

IO'

cells/ml per ampoule. Frozen cells of T-ALL patients were used for all cell cultures and for DNAisolation.Patient cells were donatedaccordingto aprotocol approvedbytheCommittee

onInvestigations InvolvingHumanSubjectsattheUniversity of

Cali-fornia,San Diego,andinformedconsent wasobtained fromthe

pa-tients ortheir parents. Peripheral blood cellswerecollectedforthe purpose ofroutineclinicaldiagnosis,and cells thatremained afterthe

diagnosticprocedureswerefrozenforfutureuse.T-ALLcellsofpatient

H.A.thatwerestudiedinthisreport wereobtained from thepatientat first relapse (see below).

Casehistory.Patient H.A. isaCaucasiangirlwhowasnoted to have

fever, malaise, andgeneralized lymphadenopathy at age 7yr 2mo.

Examination of herperipheralblood showed awhite blood cell count of 16.9X 109/literwith 90%lymphoblasts,8.8g/dl hemoglobin,and 134 x 109/literplatelets. Bone marrowaspiration showed98% blasts, which werepositivefor CD5 andCD7,andweaklypositive for CD4,

confirmingthediagnosis ofacuteTlymphoblastic leukemia. She com-pleted a2-yr courseofchemotherapy according to treatment No. 2 of

Pediatric Oncology Group protocol No. 8691. It consists of a 6-wk

induction regimen with vincristine, prednisone, cyclophosphamide,

adriamycin, cytosine arabinoside,andL-asparaginase, followedby 6-wkconsolidationwith VM-26 + cytocine arabinoside and

cyclophos-phamide+adriamycin+L-asparaginase.This was followed by 10 cy-clesof 9-wkmaintenance therapyand weekly L-asparaginase for 20 wk. She alsoreceived intrathecaltreatmentwithmethotrexate+cytosine

arabinoside+hydrocortisone for prophylaxis ofCNS leukemia. At age 9 yr 9 mo, 8moafterthecompletionofPediatric Oncology Group protocolNo. 8691 treatment, sherelapsedwithsplenomegaly

andawhite bloodcountof17.5X 109/literwith 55% blasts. Cell

sur-facemarkeranalysisrevealedsimilarimmunophenotypeas at

diagno-sis. H.A.wasthen enrolled inPediatricOncologyGroupprotocolNo. 8862 and underwent reinductiontreatmentwithvincristine,

predni-sone,daunomycin,andL-asparaginase.Atthe endofthe reinduction herperipheral blastscleared,but bonemarrowcontained 63% blasts.

Aftertwo coursesofintravenous6-mercaptopurineandmethotrexate,

shedevelopedmarrowaplasiaandstaphylococcalsepsis.Thiswas fol-lowedbyresurgenceof peripheralblasts and she died of infection 4mo

afterrelapse.

DNA extraction.Oneampoule(I07livecells)of each frozen T-ALL

patientbonemarrow orperipheralblood("primary")samplewaslysed

in500 Adoflysisbuffer (0.5%SDS, 0.1 MNaCl,50mMTrispH8.0,1 mMEDTA). 5

,d

proteinaseK(10mg/ml)wasaddedtoeachsample

andsampleswere incubated at 37°C overnight. Sampleswerethen heatedat68°C for 5 min, mixed with75MI of 8Mpotassiumacetate, extracted oncewith chloroform, precipitatedwithethanol, and dis-solved in 300Ml ofTE buffer. Thesamples werethen treated with DNAase-free RNAase, extractedagainwithchloroform, reprecipitated

with ethanol, and dissolved inafinal volumeof- 30,lTEbuffer.

DNAsequencing.Solid-phasesequencingofin vitroamplified geno-micDNA wasused, in whichgenomic DNAwasamplified byPCR

usingbiotinylated primers. I

Mg

ofgenomicDNAwasusedastemplate

ina

100-Ml

PCR reaction with 12pmol ofbiotinylatedprimerJY3 (5'-CAACCAGCCCTGTCGTCTCT-3') and 36 pmol of nonbiotiny-lated primer MH22 (5'-CTGTTCACTTGTGCCCTGAC-3').

40,Ml

of

this reactionwasincubated withmagneticbeads conjugatedcovalently

with streptavidin (Dynabeads M280-Strepavidin; Dynal, Oslo,

Nor-way)whichwereusedtoselectively immobilize the biotin-labeled PCR product and allow melting of the DNA duplex, followed by elution of the nonlabeled single strand. The immobilized single-stranded DNA was then used as sequencing template using the Sequenase (U.S. Bio-chemical Corp., Cleveland, Ohio) protocol andan internal primer,

MH26 (5'-GACTTTCAACTCTGTCTC-3'). Asymmetric PCR

ampli-fication of cDNA and direct sequencing analysis were done as

previ-ously described (13).

PCRamplificationofgenomic DNA. Two independent polymerase chainreactions were carried out for each DNA sample to analyze p53 codons 143 and 175. 1 Mg of genomic DNA was used in each 100-MA' reaction. The four PCR primers used and the reaction conditions were asdescribed ( 15).

Restriction enzyme digestion. The PCR-amplified DNA fragments weredigested with restriction enzyme HhaI, fractionated in 8% poly-acrylamide gels, stained with ethidium bromide, and photographed underultraviolet light.

Metabolic labeling andimmunoprecipitation. 5 X 106 cells were labeled for 3 h in 100 MCi/ml[35S~methionine/[35S]cysteine(translabel, ICN Biomedicals, Inc., Costa Mesa, CA). Cells were lysed in EIA buffer (250 mM NaCl, 50 mM Hepes, 0.1% NP40, 1%aprotinin,500

MM

PMSF,1mMEDTA)for 15minonice. The

lysate

was

centrifuged

at 100,000 g for 30 min and the pelletdiscarded. Equal amounts of radioactive materialwerereactedwith the specific antibodies G59-12 (Pharmingen, San Diego, CA), PAb 240 (Oncogene Science, Inc., Man-hasset, NY; 22), PAb 1620 (23), or the nonrelated (SV40 T-Ag) anti-bodyPAb419(24) for 4 h at 4°C. The immune complexes were col-lected on immobilized recombinantproteinA(Repligen Corp., Cam-bridge, MA), washed three times with EIA buffer, once with PBS, and boiled for 2 min insamplebuffer.Sampleswerethen loadedon a10%

SDS-polyacrylamide gel. Half-livesweredetermined by chasingthe radiolabeled cells forappropriatetimeperiodsinnonradioactive

me-dium, followed by immunoprecipitationasabove. The radioactivity

assignedtothep53bandswasquantitated bydirectcountingofthe gel bands inascanner,orby densitometry.

Karyotype analysis. Karyotype analysiswasdone aspreviously de-scribed ( 13).

HLA-DRanalysis.HLA-DRanalysiswasconducted by reverse slot blot PCRhybridization (25).75-bpfragmentsof HLA-DR genes were

amplified by PCR, labeled with32p, andhybridizedtoimmobilized

allele-specific oligonucleotides, encoding aminoacids 67-74 of HLA-DR 3-chain genes.Allele-specific oligonucleotidesfor six other HLA-DRB1 and three HLA-DRB3 genes wereincluded in the analysis. Primer and allele-specific oligonucleotides sequences were deduced frompublisheddata(26,27).

Results

H.A.leukemia cells haveaheterozygous pointmutation in the p53 gene. Genomic DNA of T-ALL patient samples was

screened for point mutations at two "hot spots" of thep53 gene,codons 143 and175, usingPCRanalysis.A 1 ll-bp

frag-ment surrounding p53 codon 143 and a 319-bp fragment surroundingp53codon 175 wereamplifiedanddigestedwith

the restriction enzyme HhaI. Some mutations (e.g.,

GTG-*'GCG, Val---Ala)in codon 143wouldresult incleavage

(withHhaI)of the 1Il-bpPCR-generated fragmentto a68-bp

and a43-bp fragment (13). Human placenta-derivedcontrol DNA as well aspatient sampleDNA showed the 1 I l-bp un-cleavedpattern byHhaIdigestion (datanotshown),suggesting

the absence of thisspecificmutation in codon 143.Similarly,a mutation within codon 175 would abolish anHhaI site,leading

tothe cleavage (with HhaI) of the 319-bp PCR-generated

(4)

of fourfragments(of 216 bp, 49 bp, 36 bp, and 18 bp). DNA extracted from peripheral blood nucleated cellsofrelapse T-ALLpatientH.A.showedfiverestrictionfragmentsof 216 bp, 67bp, 49bp, 36bp,and 18 bp, by HhaIdigestion(Fig. 1, lane 1), revealingthe presenceof an apparent heterozygous muta-tion at codon 175 in the peripheral blood/leukemia cells of

patientH.A.

Establishment of T cell and B cell lines from peripheral blood cells ofpatient H.A. The peripheral blood cells ofrelapse T-ALL patientH.A. contained, in addition to 55% leukemic blasts,amixture ofother cell types. The heterozygous appear-anceofthemutationatp53 codon 175 may therefore be due to amixture ofleukemiccells that possess a homozygous

muta-tionatcodon 175, and nonleukemic cells that are wild type at thatposition. Alternatively,theleukemic cells may harbor one mutatedp53 allele and one allele that is wild type at codon 175.

Finally,thepatient may harbor an inherited heterozygous

mu-tationthatispresentinall cellsofthe blood sample.

To examine the nature of the mutation, leukemic T cell

lines and an immortal B cell line were established from the same ampoule of frozen peripheralblood cells of HA. Eight

independently derivedT celllines HATL were established by

growingthe cells in the presenceof20 ng/ml recombinant IGF-I in a low oxygen (5%)tensionCO2 incubator (28, 29). The

lineswerethen cloned by end point dilution. The B cell line HABL wasestablishedbytransformingthe cellswith Epstein-Barrvirus.TheleukemicTcelllinesand theimmortal Bcell

linehave beenmaintainedin continuous culture for 2 yr, and have beenfrozen and thawed repeatedly (30). The lines share

an identical human leukocyte antigen type (DR2,7), as is

shown inFig.2,suggestingthat they wereindeed derived from thesameindividual. Thelong-termHATLlineshave the fol-lowing differentiation markers: CD3-, CD4-, CD5-, CD7' (62% ofthe cells),CD8-,CD9+ (76%), CD38+ (35%), CALLA-, and lack B cellmarkers CD19-, CD20-, and cytoplasmic Mu-. On thebasis ofcellsurfacemarkers HATLmightthus be classi-fiedas"pluripotent lymphohematopoietic"cell(31, 32).

Inter-1 2 3 4 5 6 Figure1. Detectionofamutationat

codon 175of the p53 gene in blood cellsamplesof T-ALLpatientH.A. Restriction enzyme digestion of

PCR-amplified fragmentsfrom the geno-micDNA.GenomicDNAextracted 216bp- from different cell samples of patient

H.A. wasamplifiedby PCR around thep53codon 175region. Amplified

DNAfragmentswerethendigested

67bp-

with the restriction enzyme HhaI,

49bp- fractionated on an 8%polyacrylamide

gel,stained withethidiumbromide,

36bp- andphotographed underultraviolet

light.(Lane1)Primaryperipheral bloodnucleated cellsof T-ALL pa-tientH.A.(Lane2)HATL,aclonal leukemiaTcellline grown from the

H H H peripheral blood ofpatientH.A. (Lane

36bpI49bp

I

ift

I 216bP 3)Cloned DNAsamplewith homo-wt GCGC zygous mutation at codon 175 (13).

mut GGGC (Lane 4) Wild-type DNA control from

anormal humanplacenta.(Lane5)

HABL, the immortalized B cell line grown from the peripheral blood ofpatientH.A.(Lane6) DNAsizemarker.

1 2 3 W4W13 5

S

I

*

I

I

7 8 9 2B3 3B3 7B3

1 2 3 W4W13 5

0

8 9 2

7 8 9 2B33B3 7B3

Figure2. HLA-DR

analysisofHATL and HABL cell lines. Re-HABL verseslot blot PCR

hy-bridizationwasusedon

75-bp fragmentsof HLA-DR genes that werePCRamplified, labeled with 32P, and

hybridizedto immobi-lizedallele-specific oli-gonucleotidesencoding

aminoacids 67-74 of

HATL HLA-DR

#-chain

genes.

The lines are derived from thesame individ-ual, and share the DR2,7 specificity.

estingly, the HATL leukemic lines are dependent for growth on exogenously supplied (20 ng/ml) IGF-I, and have maintained thisdependencefor 2 yr of continuous culture.Propagationof the HABL line is independent ofexogenously suppliedIGF-I. DNA was extractedboth from the HATL and HABL lines,

amplified byPCR around codon 175 anddigestedwith HhaI. Fig. 1 (lane 2) shows thatHATL cell lines areheterozygously mutated in p53 at codon 175, while the HABL cell line is wild typeatthesameposition (Fig. 1, lane 5) since it possesses the HhaIrecognition site, like the wild type control DNA shown in lane 4. Thus, the HATLleukemic cells carry aheterozygous

mutationatp53 codon 175,while theimmortalizedBcell line established from thesamepatientcarriesahomozygouswild typecodonatthisposition. Hence,theheterozygousp53

muta-tion found in the T-ALL cells ofpatient H.A. is of somatic

ratherthanhereditary origin.

Toexamine whetherthep53gene in the leukemic cells of patient H.A. harbor mutations other than the one at codon

175, wesequenced the entire cDNA of several HATL lines. RNAwasextracted fromHATLcellsand reverse transcribed

into cDNA. The full length cDNA ofHATL cellswas then amplified by PCR and directly sequenced as described in

Methods. No additional mutations were found in the p53

cDNA of HATL. FulllengthcDNA of the HABL cell line was

alsosequenced,confirming the wildtypestatusofthep53gene inthese cells. Inaddition,exon5 ofgenomicDNA fromboth

the HATLand HABL cell lines as wellas from the original

patient samplewassequencedtoverifythe Tcell

origin

ofthe

heterozygouspoint mutationatcodon 175

(CGC-*oGGC,

Fig.

3). All sampleswereheterozygous (CGC/CCC, arg/pro)for a

knownpolymorphismatcodon72(33). Thedata thus suggest that the HATL cell lines (fourhave beenanalyzed), and the

primary (in vivo)HAcellsfrom whichthelineswerederived,

harboredonlyoneheterozygouspoint mutation. Furthermore,

in vitro establishment ofthe HATLlines aswellas EBV-in-duced transformation (immortalization) of the patient's B

cells, were notassociated with the introduction ofadditional

p53 gene mutations.

(5)

A

G A T C

_ _. _W

0.=-1 8

... _

*__

-\

_-~ __o

ft

B

GA T C

Wm

=t.-__

_t-_M

_ _

_*Mb

C

G A T C

_w..

__

_ _

=

_

_ .. ._*

_

...S

_

....-. R

._ e

rs

_ rW

_ _

..., >

,, .. J

_ \

--_ C/G

_ _ _

=

-_

__

w__

-_ _

__

Figure 3. Sequenceanalysis of exon 5 of genomic DNA from HABL (A) and HATL (B) celllinesandfromprimaryperipheral blood nu-cleatedcellsof patientHA(C). DNA was extracted and amplified by PCRusingprimers JY3 and MH22 and sequenced with internal

primerMH26 (see Methods). Shown is the heterozygous mutation

foundin thep53sequence atcodon 175 inboth the patient's periph-eral blood sample and in DNA extractedfromoneof theestablished

HATL celllines.

this

antibody precipitates p53 protein

from both HATL and the HABLlines. The

p53

protein

ofHAcells have the double-bandpattern

characteristic

ofhuman cells

possessing

both

poly-morphic allelesatcodon 72(33).

Immunoprecipitation

with the monoclonal

antibody

PAb240

(Fig.

4,lanes3 and 7),

which

recognizes certain

mu-tantbutnot

wild

type

forms

of

p53

(22, 34, 35),

resulted in the

detection

oftwo mutant

p53

proteins

inHATL

lines,

butnotof thetwowildtype

p53

proteins

of the HABL

line, confirming

therestrictionenzymeand

sequencing

dataof themutantand

wild type status of

p53

in

either

celltype,

respectively.

PAb

HATL

HABL

MOLT-4

IM

l 2

3

4

115

6

7

8

9

10

11I

kd

i~

97.4

p,

69-rn.

*

-0

ow _ & d 5

Figure 4.p53proteinanalysisofHATLand HABL celllines. Cells werelabeled with

[35S]methionine/[35S]cysteine

translabel, lysed in E1Abuffer,andimmunoprecipitatedwith: (lanes I and 5)PAb419,

anonrelevantcontrolantibody; (lanes 2,6 and9) G59-12 which

rec-ognizesallforms ofp53;(lanes3,7and10)PAb240, which recog-nizes certain mutant conformationsof thep53protein; and (lanes4,

8 and11)PAb1620 which isspecificfor wild typep53.

Immunopre-cipitateswereanalyzedon a10% SDS-PAGE gel.

1620, whichrecognizesp53 proteinin the wild type conforma-tion, precipitated both proteins from the HABL cell line, but recognized only thefaster migrating protein from HATL cells (Fig. 4, lanes 4 and 8). Lanes 9-1 1 of Fig. 4 show the

immuno-precipitationofp53 from metabolicallylabeled Molt-4T-ALL

cells, which we have previously shown to express only wild type p53 (36).

RecognitionbyPAb240of bothproteinsof theHATL cell lines confirms the ability of mutant p53 protein to drive co-translatedwild type p53 into the mutant conformation, as has

been observedin vitro for cotranslatedmutant andwildtype p53proteinsbyMilnerand Medcalf(37). Recognition by PAb

1620 ofonly thefaster migrating, codon 72"-containing pro-teinfrom HATLlinessuggeststhat HATL cells synthesize both wild type and mutant p53 proteins.

Analysis of the metabolic stability ofthep53proteins in the HATL and HABL cells by pulse-chase experiments (not

shown) indicates that both p53 proteins inHATLcells decay

with a half-life of4.2 h, while the wild type formofp53 in HABLcells has ahalf-life of 1.2 h. As a comparison, in our

hands, p53 protein in activated (IL-2 grown) normal human

peripheral bloodTcellsturns overwithahalf-life of 0.5h (36). Karyotype ofHA TL and HABLcell lines. Karyotype

analy-sis showed that the HABL cells possessed a normal diploid

karyotype,whilethedifferentHATLisolates had several

abnor-malities, specifically a clonal rearrangement of chromosome

lp,monosomy 7, or a rearranged chromosome 7, but normal

chromosomes 17(thep53gene maps tohuman chromosome

17p13). Fourexamples of karyotypes ofHATL cellsare shown

inFig. 5.

Discussion

Severallines of evidence suggest that thedevelopmentof

lym-phoid

and other

hematopoietic

neoplasia

in humans is

asso-ciated with alterations of the

p53

gene. The presence of

high

levels of

p53 protein,

which ischaracteristic oftumors harbor-ingmutated

p53 alleles,

has been documentedinsomehuman

lymphoproliferative

disorders

(38,

39),

and in blastcrisis CML

(9, 10, 40, 41). In Tcell leukemias thepresenceof

p53

muta-tions has been documented

by

us

(13,

and this

paper),

and

others

(14, 19),

though

someauthors failedto find any such

mutations

(21, 42)

in

diagnosis

T-ALLcells.

6of11 human T leukemia cell lines thatwehave studied

possessed

independent

mutations of

p53 alleles,

prompting

the

question

whether the mutations foundinthese lines may be

associated with their establishment rather than

being

asso-ciated withthe disease in vivo. This

question

was

particularly

acute

following

the report that establishment of

long-term

cul-turesofratembryo fibroblastsisassociatedwith the induction

of

p53

mutations

(20). Hence,

conceivably, p53

mutations

found inhuman Tleukemia linesmayalso havebeen induced

during

invitroestablishment.

The present

study

was

designed

toexamine whether

p53

mutations are associated with some T-ALL cases in

vivo,

whetherthemutationsareretainedincelllines

developed

from the

leukemias,

and whether establishment of T leukemia cell

lines is associated withtheinductionof mutationsin

p53.

Ex-tensive

study

of the T-ALL

relapse patient

H.A.nowsuggests

that

(a)

leukemia cells taken from T-ALL

patient

H.A.

pos-sesseda

heterozygous

mutationat

p53

codon

175; (b)

the

(6)

Ads'7

*;.,>k I.

b!

tis

to

,

*V* "M q#0

4

it

- *

o

/

B A

* tt

4*h?1

(

'9

IV-"

X.

A.

c

D

Figure 5.Karyotypes ofHATLcells. Fourmetaphase spreadsshow thetypicalchromosomeabnormalities observed in HATL cells. All cells

an-alyzed haveastructurally rearrangedchromosome No. 1(arrowheads)and monosomyofchromosome No.7,or(D)arearrangedchromosome No.7(thin arrows).Inaddition,variousother,occasional abnormalities have beenobserved,includingtrisomyof chromosome No. 18(A),and unidentified marker chromosomes(thickarrowsinA and D).Theabnormalitiesshownwerepresent in alleightHATLcelllinesstudied. The

immortalizedBcell line HABL hasanormaldiploid karyotypeandisnotshown.

the

peripheral

blood of the

patient; (c)

establishment ofthe

immortalized (normal) B cellline HABLwas not associated withtheinductionof mutations in

p53; (d)

establishment ofT

leukemia cell lines HATL was notassociated with the induc-tionof additional

p53

mutations; (e)

the

p53

mutation in

re-lapseT-ALL patientH.A. wasof somatic origin; and

(f

)

pa-tient H.A.'s leukemicTcellsresemblethe Tleukemialines that

wehave studied previously (13) withrespect tothe heterozy-gous natureof the p53

mutations,

aswellaswithrespect

of

the absence

of

lossof

heterozygosity.

Our resultsdiffer from those of Gaidanoetal.

(21)

andof

Jonveaux

(42),

whosuggested that

p53

mutationsare not im-plicated in the natural

history

of T-ALL. These

ostensibly

dis-cordantsetsof data maybe resolved in severalways. On the

one hand, Gaidano and Jonveaux studied T-ALL

diagnosis

samples

(i.e.,

samples

from

early, nontreated cases) which ap-pear not to possess p53 mutations, while p53 mutations are

associatedwith relapseT-ALL(thispaper, and14,19). Further-more,establishment ofT-ALLcelllineshas until recently been

an

infrequent

accomplishment (43, 44), andestablishment of

T-ALLlinesmay have selectedfor samples harboring

p53

mu-tations. Indeed, until recently establishment ofT-ALLlines

hasbeenlimitedtorelapsecases(45), hencethehighfrequency of

p53

mutationsinestablishedcelllines mayreflectthe situa-tion in relapse cases only. The most reasonable way to

recon-ciletheavailabledataistosuggest thatp53 mutationis

infre-quently associated withT-ALL

"diagnosis" disease,

but is al-teredduringrecurrence of thedisease; casesthat possessp53

mutations have a selectiveadvantageofbecomingestablished

cell lines. This

interpretation

of the availabledata is in

accor-dance withrecentevidence

using single-strand

conformation

polymorphism and direct sequencing analysis ofperipheral blood-and bone marrow-derived leukemia

samples

takenat

diagnosisand atrelapse. These results indicatethat mutations

of p53are

predominantly

associated with the relapse

phase

of

the disease(Hsiao, M.,J.Yeargin, E. Dorn,A. L.Yu, andM.

Haas,manuscriptinpreparation).

Theleukemic cells of patientH.A. werefoundtopossess a

heterozygouspoint mutationatcodon 175

(Arg-*~Gly).

Codon 175

mutations

are amongthemore potently oncogenic p53

mutations, possibly

evenin thepresenceof expressed wildtype

p53 alleles (46). Thesuppressive action ofthe wildtypealleleis thoughttobebridled by the mutant allele via a dominant-nega-tivemechanism. Hence,theheterozygouscodon 175 mutation

foundin H.A.'sleukemiacells mayhavegiven thecellsa dis-tinct growth advantageinspiteofthe presenceofanexpressed wildtype allele (47).Interestingly,in HATL cellstheproductof themutantallele and themajority oftheproduct ofthewild

type allele possess a mutantimmunophenotypeon immuno-precipitation gels (Fig. 4; in HATL cells the products of the

(7)

atcodon72,asthetwop53 allelescarry aprolineor anarginine

atthisposition,respectively (33).

InHATLcells, theacquisition by the wildtypep53protein of themutant immunophenotype under the influence of the

mutantp53 proteinmay represent a

mechanism for

the

func-tionalinactivation of

p53

bya

dominant-negative

mechanism (37). This isreminiscent of the data presented by

Milner

and Medcalf (37) who showed thatupon

cotranslation

in

vitro of

mutantandwildtypep53 alleles themutantp53can

influence

theconformation of the cotranslated wildtype

p53

anddrive it into themutantimmunophenotype. Imposition of themutant

immunophenotype onthewild type geneproduct is brought aboutby the formation ofp53 wild type/mutant complexes,as wasdocumented by Milner(32).Thusthe resultsof immuno-precipitation of

metabolically

labeled

p53

inHATLcellsagrees

withtheinvitro data of Milner and Medcalfonthefunctional

inactivation of p53

genes

by

a

dominant-negative

mechanism. Themutation in onealleleof the

p53

geneinHATLcells confersa mutant

immunophenotype

onmuch

of

thewildtype

protein, encoded by the normal, nonmutated allele. However,

asis shown by the karyotype data, mutation

of p53

isnotthe only

abnormality

inH.A.'sleukemic

cells,

andabnormalities in chromosome No. 1 and No. 7 have been found. Indeed, since

carcinogenesis

is a

multistep

process, mutation in

p53

would not byitself be expectedto

confer

malignancy.

Evidence forthis notion is amply

provided

by thestateof

p53

in individ-ualsof Li-Fraumeni Syndrome families

(17,

18).

Interestingly,

abnormalities in chromosomes Nos. 1,

7,

and 17 havebeen

associated

with

refractoriness

to

chemotherapy

and poor

sur-vival in human lymphomas and neuroblastomas

(48, 49).

Preliminary

evidence suggests that mutation of the

p53

gene is

associated

with the recurrence of T-ALL,

just

asit is

associated with

the

progression of

the

tumorigenic phenotype

of other humantumors. Induction

of

remission ofT-ALL

by

chemotherapy ismoredifficult with

increasing

numbers of

re-lapse

episodes.

Mutation of the

p53

gene thereforeappearsto

point

to apoor

prognosis

of

T-ALL,

andonewouldexpectthat

some

specific p53 mutations

wouldpresenta worse

prognosis

than others. Itwouldbe

important

todetermine the

relation-ship

betweendifferent

p53

mutationsandthe

prognosis

of the disease. Itwould also be

important

toattempttosuppressthe

effects of

mutated

p53 by

the

introduction

of

properly-pro-moted wildtype

p53

constructs

(50).

This

laboratory

is

actively

seeking

answersin both of theseareas

(30).

Acknowlednments

This workwassupportedinpartbygrants from the American Cancer

Society (CH456), the US Department of Energy (DE-FG03-91

ER61171, and the National Cancer Institute,National Institutes of Health(ROCA56075,andU1OCA28439 [toA. L. Yu]),US

Depart-mentof Health,Education,andWelfare.

References

1.Finlay,C. A.,P. W.Hinds,andA.J.Levine. 1989. Thep53proto-oncogene

can act as asuppressoroftransformation.Cell.57:1083-1093.

2.Nigro,J.M.,S.J.Baker,A.C.Preisinger,J. M.Jessup,R.Hostetter,K.

Cleary,S.H.Bigner,N.Davidson,S.Baylin,P.Devilee,etal.1989.Mutations in

thep53geneoccurindiverse humantumourtypes.Nature(Lond.). 342:705-708.

3. Iggo, R.,Gatter,J.Bartek, D. Lane,and A. L. Harris. 1990. Increased

expressionofmutantformsofp53oncogeneinprimarylungcancer.Lancet.

335:675-679.

4.Takahashi, T., M. M. Nau,I.Chiba,M.J.Birrer,R.K. Rosenberg, M. Vinocour, M.Levitt, H. Pass, A. F. Gazdzr, and J. D. Minna. P53: a frequent target forgenetic abnormalities in lung cancer. Science (Wash. DC). 246:491

-494.

5.Bressac, B., M. Kew, J. Wands, and M. Ozturk. 1991. Selective G to T

mutations ofp53gene inhepatocellularcarcinoma fromSouthernAfrica.Nature

(Lond.).350:429-431.

6. Hsu, I.C.,R.A.Metcalf,T.Sun, J.A.Welsh, N. J. Wang, and C. C. Harris. 1991.Mutationalhotspot in thep53gene in humanhepatocellular carcinomas. Nature(Lond.). 350:427-428.

7.Sidransky, D.,A.VonEschenbach,Y.C.Tsai,P.Jones, I.Summerhayes,

F.Marshall, M.Paul,P.Green,S. R.Hamilton,P. Frost,and B. Vogelstein. 1991.

Identification ofp53gene mutations in bladder cancers and urine samples. Science(Wash.DC).252:706-709.

8.Marks,J.R.,A.M.Davidoff,B. J.Kerns, P.A.Humphrey, J. C. Pence, R. K.Dodge, D. L.Clarke-Pearson,J. D.Iglehart, R. C. Bast, and A. Berchuck. 1991.Overexpressionandmutationofp53inepithelialovarian cancer. Cancer Res.51:2979-2984.

9.Ahuja,H., D.Bar-Eli,S. H.Advani,S.Benchimol,and M. J.Cline. 1989. Alterations in thep53gene and the clonal evolution of theblastcrisis of CML. Proc.Nat!.Acad.Sci. USA. 86:6783-6787.

10.Kelman, Z.,M.Prokocimer,S.Peller,Y.Kahn,G.Rechavi,Y.Manor,A.

Cohen,and V. Rotter. 1989.Rearrangements inthep53gene inPhiladelphia

chromosomepositive chronic myelogenous leukemia. Blood. 74:2318-2324.

11.Diller,L., J.Kassel,C. E.Nelson,M.A.Gryka,G.Litwak,M.Gebhardt,

B.Bressac, M.Ozturk,S. J.Baker,B.Vogelstein,andS. H.Friend. 1990.P53 functions as a cell cyclecontrol proteinin osteosarcomas. Mol. Cell. Biol. 10:5772-5781.

12. Masuda, H. H., C.Miller,H. P.Koeffler,H.Battifora,andM.J.Cline. 1987. Rearrangementofthep53gene in humanosteogeniccarcinomas. Proc.

Nat!.Acad.Sci. USA. 84:7716-7719.

13.Cheng,J., andM. Haas. 1990. Frequent mutations in thep53tumor

suppressor gene in humanleukemiaT-celllines. Mol.Cell.Biol. 10:5502-5509. 14.Felix,C. A., M. M.Nau,T.Takahashi,T.Mitsudomi, I. Chiba,D.G.

Poplack,G. H.Reaman,D. E.Cole,J. J.Letterio,J.Whang-Peng,etal. 1992.

Hereditaryandacquiredp53gene mutations inchildhoodacutelymphoblastic leukemia. J.Clin.Invest.89:640-647.

15.Baker,S.J.,E. R.Fearon,J. M.Nigro,S. R.Hamilton,A.C.Preisinger,

J. M.Jessup,P.vanTuinen,D. H.Ledbetter,D. F.Barker,Y.Nakemura,etal. 1989. Chromosome 17deletionsandp53 gene mutations in colorectal

carci-nomas.Science(Wash. DC).244:217-221.

16.Shirasawa, S.,K.Urabe,Y.Yanagawa,K.Toshitani,T.Iwama,and T.

Sasazuki. 1991. P53 gene mutations in colorectaltumors frompatientswith

familialpolyposiscoli.Cancer Res. 51:2874-2878.

17.Malkin, D.,F. P.Li,L.C.Strong,J. F.Fraumeni, Jr.,C. E.Nelson,D. H.

Kim,J.Kassel,M. A.Gryka,F. Z.Bischoff, M.A.Tainsky,and S. H. Friend. 1990. Germ linep53mutations inafamilialsyndromeof breast cancer,

sarco-mas, and otherneoplasms.Science(Wash.DC).250:1233-1238.

18.Srivastava, S.,Z.Zou,K.Pirollo,W.Blattner,and E. H.Chang.1990.

Germ-linetransmission ofamutatedp53gene inacancer-pronefamilywith Li-Fraumenisyndrome.Nature(Lond.).348:747-749.

19. Felix,C. A., D. D'Amico, T.Mitsudomi,M. M. Nau,F. P.Li,J. F.

Fraumeni,D. E.Cole,J.McCalla,G. H.Reaman,J.Whang-Peng,etal. 1992. Absence ofhereditarymutations intenfamilialleukemiapedigrees.J.Clin.

In-vest.90:653-658.

20.Harvey,D.M.,andA.J.Levine. 1991. P53alterationisa common event

in the spontaneous immortalization ofprimaryBALB/c murineembryo fibro-blasts.Genes & Dev. 5:2375-2385.

21.Gaidano,G.P.,Ballerini,J. Z.Gong,G.Inghirami,A.Neri,E. W.

New-comb,I.T.Magrath,D. M.Knowles,and R. Dalla-Favera. 1991. P53mutations

in human lymphoid malignancies: association with Burkitt lymphoma and

chroniclymphocyticleukemia. Proc.Natl. Acad.Sci. USA.88:5413-5417.

22.Gannon,J.V., R.Greaves,R.Iggo,andD.P. Lane. 1990.Activating

mutations inp53producea commonconformational effect.Amonoclonal

anti-bodyspecificfor themutantform. EMBO(Eur.Mol. Biol.Organ.)J. 9:1595-1602.

23.Ball,R.K.,B.Siegl,S.Quelhorst,G.Brandner,andD.G. Braun.1984. Monoclonal antibodiesagainstsimian virus 40 nuclearlargeTtumorantigen:

epitopemapping, papovaviruscross-reaction and cellsurfacestaining.EMBO

(Eur.Mol. Biol.Organ.)J. 3:1485-1491.

24.Harlow,E.,L. V.Crawford,D.C.Pim,and N.M.Williamson. 1981. Monoclonal antibodiesspecificfor SV40tumorantigens.J. Virol. 39:861-869.

25.Saiki,R.K.,P.S.Walsh,C. H.Levenson,and H.A.Erlich.1989.Genetic

analysisofamplifiedDNAwith immobilizedsequence-specificoligonucleotide

probes.Proc.Nat!.Acad.Sci. USA.86:6230-6234.

26.Bell,J.I.,D.Denney,L.Foster,T.Belt,J. A.Todd,andH.0.McDevitt. 1987.Allelic variation inthe DRsubregionofthehumanmajor

histocompatibil-ity complex.Proc.Natl.Acad.Sci. USA.84:6234-6238.

(8)

Maccari, D. Goldberg, H. Murphy, J. Schwenzer, et al. 1986. Molecular diversity

ofHLA-DR4haplotypes. Proc. Natl.Acad.Sci. USA. 83:2642-2646. 28.Gjerset,R., J.Yeargin,S. K. Volkman, V. Vila, J. Arya, and M. Haas. 1990.Insulin-like growthfactor-I supports proliferation ofautocrine thymic lym-phoma cells with a pre-T phenotype. J.Immunol. 145:3497-3501.

29.Smith, S. D., P. McFall,R.Morgan, M.Link, F. Hecht, M. Cleary, and J. Sklar. 1989. Long-term growth of malignant thymocytes in vitro. Blood. 73:2182-2 187.

30. Cheng, J., J.-K. Yee, J. Yeargin, T. Friedman, and M. Haas. 1992. Sup-pression of acutelymphoblastic leukemia by the human wild-type p53 gene. Cancer Res.52:222-226.

31. Kurtzberg, J., S.H.Bigner, and M. S. Hershfield. 1985. Establishment of theDU.528 humanlymphohemopoietic stem cell line. J. Exp.Med. 162:1561-1578.

32. Kurtzberg, J., T. A. Waldmann, P. Davey, S. H. Bigner, J. 0. Moore, M. S. Hershfield, and B. F. Haynes. 1989.CD7',CD4-, CD8- acute leukemia: a syn-dromeofpluripotentlymphohematopoieticcells.Blood.73:381-390.

33.Harris, N., E. Brill, 0. Shohat, M. Prokocimer, D. Wolf, N. Arai, and V. Rotter. 1986. Molecular basis for heterogeneity of the human p53 protein. Mol.

Cell. Biol.6:4650-4656.

34.Lane, D. P., and L. V.Crawford. 1979.T-antigen is bound to a host protein inSV40-transformed cells. Nature(Lond.).278:261-263.

35. Varley, J. M., W. J. Brammar, D. P. Lane, J. E. Swallow, C. Dolan, and R. A.Walker.1991. Loss of chromosome 1 7p 1 3 sequences andmutation of p53

in humanbreast carcinomas. Oncogene. 6:413-421.

36.Yeargin, J., J. Cheng, and M. Haas. 1992. Roleofthep53 tumor suppres-sorgene in thepathogenesis and in thesuppression ofacutelymphoblasticT-cell leukemia.Leukemia(Basingstoke).6(Suppl.3):85s-91s.

37.Milner, J., and E. A. Medcalf. 1991. Cotranslation of activated mutant p53with wild type drives the wild type p53 protein into the mutant conforma-tion.Cell.65:765-774.

38.Koeffler,H.P., C.Miller,M.A.Nicolson,J.Ranyard, andR. A. Bossel-man.1986. Increasedexpression of p53 protein in human leukemia cells. Proc.

Natl.Acad.Sci. USA. 83:4035-4039.

39.Prokocimer,M., M.Shaklai,H.BenBassat, D.Wolf,N.Goldfinger,and V. Rotter. 1986.Expression of p53 in humanleukemiaandlymphoma. Blood.

68:113-118.

40.Feinstein,E., G.Cimino,R. P.Gale, G. Alimena, R. Bertier, K. Kishi, J. Goldman, A. Zaccaria,A.Berrebi, X. P. Mental, et al. 1991. P53 in chronic myelogenousleukemia inacute phase. Proc. Natl. Acad. Sci. USA. 88:6293-6297.

41.Mashal, R., M. Shtalrid, M. Talpaz, H. Kantarjian, L. Smith, M. Beran, A. Cork, J.Trujilo,J.Gutterman, and A. Deisseroth. 1990. Rearrangement and expression of p53 in the chronic phase and blast crisis of chronic myelogenous leukemia. Blood. 75:180-189.

42. Jonveaux, P., and R. Berger. 1991.Infrequentmutations in the p53 gene in primary human T-cell acute lymphoblastic leukemia. Leukemia

(Basing-stoke).5:839-840.

43.Hirose, M., K. Minato, K. Toninai, M. Shimoyama, S. Watanabe,T.Abe, andK.Deura. 1983.Twonovel cultured celllines, A3/KAWAKAMI and A3/ FUKUDA, derived frommalignant lymphomaof B(non-T)-cellnatureofthe gastrointestinal tract. Gann. 74:106-115.

44.Huang, C.C., Y. Hou, L. K. Woods, G. E. Moore, and J.Minowada.1974.

Cytogeneticstudy of humanlymphoidT-celllinesderived from lymphocytic

leukemia. J.Natl.Cancer Inst. 53:655-658.

45. Haas, M., A. Yu, and R.Gjerset. 1990. Characteristics ofthe leukemic cell inchildhoodacutelymphoblasticTcellleukemiaatdiagnosis. Leukemia (Basing-stoke).4:230-234.

46.Hinds,P. W., C.A.Finlay,R. S.Quartin, S.J.Baker,E. R.Fearon,B. Vogelstein, and A. J. Levine. 1990. Mutantp53DNAclonesfrom human colon

carcinomascooperate withrasintransforming primaryratcells:acomparisonof the "hotspot" mutantphenotypes.CellGrowth&Differ. 1:571-580.

47.Chen,P. L.,Y.Chen,R.Bookstein,and W. H. Lee.Geneticmechanisms of tumor suppression by the human p53 gene. 1990. Science (Wash. DC).

250:1576-1580.

48.Cabanillas,F., S. Pathak, G.Grant,F. B.Hagemeister,P.McLaughlin,F. Swan, M.A.Rodriguez,J.Trujillo,A.Cork,J. J.Butler,etal.1989.

Refractori-nesstochemotherapy and poor survival related toabnormalitiesofchromosomes

17and7in lymphoma.Am.J.Med.87:167-172.

49.Brodeur, G. M., and R.L.Saylors III. 1991.Neuroblastoma, retinoblas-toma, and brain tumors in children. Curr.Opin. Oncol.3:485-496.

50. Mercer,W.E.,M. T.Shields,M.Amin,G. J.Sauve,E.Appella,J.W.

Romano, and S. J.Ullrich. 1990.Negative growthregulationinaglioblastoma

Figure

Updating...