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.
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 […]
Find the latest version:
Cell Lymphoblastic Leukemia Is of Somatic Origin and Is
Lymphoblastic Leukemia Cell
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
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
mutations, cDNAs ofthe HATL and HABL lines were
se-quenced.The HATL lines retained thesameheterozygousp53 mutation thatwaspresent in the
HABLline lacked p53 mutations.
specific anti-p53antibodies showedthat HATL cells
p53 proteins ofmutantand wildtypeimmunophenotype, while
the HABL line
synthesizedonly wild-type p53 protein. The HATLcellshadanabnormalkaryotype, while theHABL cells
diploidkaryotype.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
cate-gory of relapseT-ALL
patientsin whoma secondremission
could not be induced. (J. Clin. Invest. 1993.
lymphoblasticleukemia relapse*tumorsuppressor gene *
es-tablishment ofleukemia lines*somatic mutation of
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.
deletion,orrearrangementhave been found inawiderange
thep53genehas been demonstrated in human carcinomas
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
carcinomatousstate,ithasbeensuggested that p53
deletion isalateeventin the development ofcancer(15). How-ever,
mutationsinp53 have recently been found in
of patientswith familial
sug-gesting that p53 mutationmay alsooccurasanearlyeventin
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
Inrelapse leukemicTcelllines, both alleles have frequently been foundtobe
independentlymutated, rather thanoneallele
beingmutated, the other deleted (15, 13).Aswehaveproposed
previously (13),thehigh frequency of mutation of all alleles
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
p53inTleukemia celllines may be due to the
oflines fromT-ALLcells that already
p53mutations in vivo. Presumably, p53 mutationsare presentina
minorityof leukemiacases(21, 14, 19), but appear
inalarge fraction ofTleukemiacelllinesdue tothe advantage
sce-nariothemutationsobservedinTleukemia lines would have
originatedin vivo. Athird possibility is thatin T-ALL, p53
mutation isnot generally found in
(21), but is associated withthe relapse phase, as has been shown
Toexaminewhetherp53mutationsplay a role in the
mor-bidity ofT-ALL,weexploredtwomutation"hotspots" in the
(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
©TheAmerican Society for Clinical Investigation,Inc.
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.
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
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
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.
shedevelopedmarrowaplasiaandstaphylococcalsepsis.Thiswas fol-lowedbyresurgenceof peripheralblasts and she died of infection 4mo
DNA extraction.Oneampoule(I07livecells)of each frozen T-ALL
in500 Adoflysisbuffer (0.5%SDS, 0.1 MNaCl,50mMTrispH8.0,1 mMEDTA). 5
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
100-MlPCR reaction with 12pmol ofbiotinylatedprimerJY3 (5'-CAACCAGCCCTGTCGTCTCT-3') and 36 pmol of nonbiotiny-lated primer MH22 (5'-CTGTTCACTTGTGCCCTGAC-3').
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
MMPMSF,1mMEDTA)for 15minonice. The
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).
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
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
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
67bp-with the restriction enzyme HhaI,
49bp- fractionated on an 8%polyacrylamide
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
iftI 216bP 3)Cloned DNAsamplewith homo-wt GCGC zygous mutation at codon 175 (13).
mut GGGC (Lane 4) Wild-type DNA control from
HABL, the immortalized B cell line grown from the peripheral blood ofpatientH.A.(Lane6) DNAsizemarker.
1 2 3 W4W13 5
7 8 9 2B3 3B3 7B3
1 2 3 W4W13 5
08 9 2
7 8 9 2B33B3 7B3
analysisofHATL and HABL cell lines. Re-HABL verseslot blot PCR
75-bp fragmentsof HLA-DR genes that werePCRamplified, labeled with 32P, and
hybridizedto immobi-lizedallele-specific oli-gonucleotidesencoding
aminoacids 67-74 of
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
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
heterozygouspoint mutationatcodon 175
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.
G A T C
_ _. _W
GA T C
G A T C
_ .. ._*
,, .. J
_ _ _
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
antibody precipitates p53 proteinfrom both HATL and the HABLlines. The
proteinofHAcells have the double-bandpattern
poly-morphic allelesatcodon 72(33).
Immunoprecipitationwith the monoclonal
(Fig.4,lanes3 and 7),
(22, 34, 35),resulted in the
proteinsof the HABL
wild type status of
-0ow _ & d 5
Figure 4.p53proteinanalysisofHATLand HABL celllines. Cells werelabeled with
[35S]methionine/[35S]cysteinetranslabel, 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
Severallines of evidence suggest that thedevelopmentof
neoplasiain humans is
asso-ciated with alterations of the
p53gene. The presence of
p53 protein,which ischaracteristic oftumors harbor-ingmutated
p53 alleles,has been documentedinsomehuman
39),and in blastcrisis CML
(9, 10, 40, 41). In Tcell leukemias thepresenceof
muta-tions has been documented
thoughsomeauthors failedto find any such
6of11 human T leukemia cell lines thatwehave studied
questionwhether the mutations foundinthese lines may be
associated with their establishment rather than
asso-ciated withthe disease in vivo. This
followingthe report that establishment of
long-termcul-turesofratembryo fibroblastsisassociatedwith the induction
found inhuman Tleukemia linesmayalso havebeen induced
mutations are associated with some T-ALL cases in
leukemias,and whether establishment of T leukemia cell
lines is associated withtheinductionof mutationsin
studyof the T-ALL
(a)leukemia cells taken from T-ALL
*V* "M q#0
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.
peripheralblood 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
re-lapseT-ALL patientH.A. wasof somatic origin; and
pa-tient H.A.'s leukemicTcellsresemblethe Tleukemialines that
wehave studied previously (13) withrespect tothe heterozy-gous natureof the p53
Our resultsdiffer from those of Gaidanoetal.
p53mutationsare not im-plicated in the natural
historyof T-ALL. These
dis-cordantsetsof data maybe resolved in severalways. On the
one hand, Gaidano and Jonveaux studied T-ALL
fromearly, 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
infrequentaccomplishment (43, 44), andestablishment of
T-ALLlinesmay have selectedfor samples harboring
mu-tations. Indeed, until recently establishment ofT-ALLlines
hasbeenlimitedtorelapsecases(45), hencethehighfrequency of
p53mutationsinestablishedcelllines 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
interpretationof the availabledata is in
polymorphism and direct sequencing analysis ofperipheral blood-and bone marrow-derived leukemia
diagnosisand atrelapse. These results indicatethat mutations
predominantlyassociated with the relapse
the disease(Hsiao, M.,J.Yeargin, E. Dorn,A. L.Yu, andM.
Theleukemic cells of patientH.A. werefoundtopossess a
heterozygouspoint mutationatcodon 175
mutationsare amongthemore potently oncogenic p53
mutations, possiblyevenin 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
atcodon72,asthetwop53 allelescarry aprolineor anarginine
InHATLcells, theacquisition by the wildtypep53protein of themutant immunophenotype under the influence of the
mutantp53 proteinmay represent a
dominant-negativemechanism (37). This isreminiscent of the data presented by
Milnerand Medcalf (37) who showed thatupon
mutantandwildtypep53 alleles themutantp53can
theconformation of the cotranslated wildtype
p53anddrive 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
withtheinvitro data of Milner and Medcalfonthefunctional
inactivation of p53genes
dominant-negativemechanism. Themutation in onealleleof the
p53geneinHATLcells confersa mutant
protein, encoded by the normal, nonmutated allele. However,
asis shown by the karyotype data, mutation
of p53isnotthe only
cells,andabnormalities in chromosome No. 1 and No. 7 have been found. Indeed, since
multistepprocess, mutation in
would not byitself be expectedto
malignancy.Evidence forthis notion is amply
p53in individ-ualsof Li-Fraumeni Syndrome families
abnormalities in chromosomes Nos. 1,
7,and 17 havebeen
sur-vival in human lymphomas and neuroblastomas
Preliminaryevidence suggests that mutation of the
associatedwith the recurrence of T-ALL,
of other humantumors. Induction
chemotherapy ismoredifficult with
episodes.Mutation of the
specific p53 mutationswouldpresenta worse
than others. Itwouldbe
prognosisof the disease. Itwould also be
seekinganswersin both of theseareas
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
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