rev bras hematol hemoter. 2016;38(4):368–370
w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Letter
to
the
Editor
Differential
profile
of
CDKN1A
and
TP53
expressions
in
bone
marrow
mesenchymal
stromal
cells
from
myeloid
neoplasms
DearEditor,
Attention has been increasing focused on the role of the bone marrow microenvironment in the pathogenesis and progression of hematological malignancies, includ-ing myelodysplastic syndromes (MDS) and acute myeloid leukemia(AML).1–3 Thus,the identificationofgenesor
pro-teinsthataredifferentiallyexpressedintheabnormalbone marrownichemayprovidenewtherapeuticopportunitiesand perspectives on the biology of these diseases. In a recent report by Fei et al.,4 bone marrow mesenchymal stromal
cells (BMMSCs) from MDS patients presentedan upregula-tion ofp21and p53expressions,suggesting thatactivation ofthis pathway may contribute to the senescentbehavior of these cells.4 In contrast, another study that also
ana-lyzedthe expressionprofileofsenescence-relatedgenes in BMMSCs from MDS and healthy donorsobserved a down-regulationofCDKN1Aexpression,butnomodulationinTP53
expression.5
To provide additional evidence on p21 and p53 expres-sion inMDS BMMSCs, weverified the expressionof these genesinacohort ofeighthealthydonorswithmedianage of 45 years (range: 28–57), 23 MDS patients with median age of 70 years (range: 16–90), seven AML patients with myelodysplasia-relatedchanges(AML-MRC)withmedianage of69years(range:30–86)and12denovoAMLcases accord-ing to the WHO 2008 classification, with median age of 61years (range: 44–82).6 TheMDS groupwas comprisedof
onerefractorycytopenia with unilineagedysplasia (RCUD), four refractory anemia with ringed sideroblasts (RARS), 12 refractorycytopeniawithmultilineagedysplasia(RCMD),two refractory anemia with excess blast-1 (RAEB-1) and four refractoryanemia with excess blast-2 (RAEB-2). Bone mar-rowmononuclearcellswereisolatedbyFicoll-Hypaqueplus density-gradientcentrifugation(GEHealthcare,Uppsala, Swe-den) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and
maintainedat37◦C,normoxiawith5%CO
2.Afterthefourth passage,allpatient andcontrol-derivedBMMSCspresented a homogeneous cell population (negative for CD31, CD34, CD45andHLA-DR,andpositiveforCD73,CD90andCD105), confirmingtheirmesenchymalorigin,accordingtothe Inter-national SocietyforCellularTherapy.7 Next,thesesamples
wereusedforgeneexpressionanalysisbyquantitative poly-merasechainreaction(qPCR)inaABI7500SequenceDetection System (Applied Biosystem, Foster City, CA, USA) using specific primers forCDKN1A(p21– FW: TGTCACTGTCTTG-TACCCTTGT; RV: GCCGGCGTTTGGAGTGGTAG), TP53 (p53 –
FW: GGCGCACAGAGGAAGAGAAT; RV:
GGAGAGGAGCTGGT-GTTGTTG), and HPRT1(FW:GAACGTCTTGCTCGAGATGTGA;
RV:TCCAGCAGGTCAGCAAAGAAT).Therelativegene expres-sionwascalculated usingtheequation,2−CT.8 Statistical
analyseswereperformedbyANOVAandBonferronipost-test usingGraphPadPrism5(GraphPadSoftware,Inc.,SanDiego, CA,USA);ap-value<0.05wasconsideredstatistically signifi-cant.
In our cohort, BMMSCs from de novo AML presented increasedCDKN1AmRNAlevelscomparedtohealthydonors, MDS and AML-MRC patients (p-value <0.05; Figure 1A).
TP53 expressionwasalsohigher inde novoAMLcompared to healthy donors and AML-MRC patients (p-value <0.05; Figure1B).However,weobservednodifferencesinCDKN1A
and TP53 mRNA levels in BMMSCs from MDS and
AML-MRC patients compared to healthy donors (p-value >0.05; Figure 1). When MDS patients were stratified by the WHO 2008 classification, no differences were observed between RCUD/RARS/RCMD, RAEB-1/RAEB-2, AML-MRC and healthy donors(p-value>0.05;Figure1).
ExistingdataonMDS-derivedBMMSCbiologyare some-what controversial, reflecting differences in methodologies and the heterogeneity ofpatients.9 There is evidence that
differentculturemediacansignificantlyinfluencethe pheno-typeofmesenchymalcells.10,11Thus,thelackofoptimization
rev bras hematol hemoter. 2016;38(4):368–370
369
3.5
A
B
Healthy donor MDS RCUD/RARS/ RCMD
RAEB-1/RAEB-2AML-MRC de novo AML Healthy donor MDS RCUD/RARS/ RCMD
RAEB-1/RAEB-2 AML-MRC de novo AML *
*** ***
***
*** *
*
6
5
4
3
2
1
0
Relativ
e le
vels of
CDKN1A
mRNA e
xpression
CDKN1A expression in bone marrow mesenchymal stromal cells
TP53 expression in bone marrow mesenchymal stromal cells
Relativ
e le
vels of
TP53
mRNA e
xpression
3.0
2.5
2.0
1.5
1.0
0.5
0.0
n=08 n=23 n=17 n=06 n=07 n=12 n=08 n=23 n=17 n=06 n=07 n=12
Figure1–CDKN1AandTP53expressioninbonemarrowmesenchymalstromalcellsfrommyelodysplasticsyndromes,
acutemyeloidleukemiawithmyelodysplasia-relatedchanges,denovoacutemyeloidleukemiaandhealthydonors.
QuantitativepolymerasechainreactionanalysisofCDKN1A(A)andTP53(B)mRNAexpressioninbonemarrow
mesenchymalcells.TheHPRT1genewasusedasanendogenouscontrolandahealthydonorwasusedasacalibrator
sample.Horizontallinesindicatemedians.*p-value<0.05,**p-value<0.01,***p-value<0.001;ANOVAtestandBonferroni post-test.
specificmarkersinBMMSCstudies.IntheprotocolusedbyFei etal.,4BMMSCswereculturedinaspecialhuman
mesenchy-malstemcellgrowthmedium,whilePavlakietal.5cultured
BMMSCsusingDMEM.
WithregardtotheCDKN1AandTP53expressionprofilein
denovoAMLBMMSCs,ourresultscorroboratethefindingsof Ruvoloetal.,12whoobservedanupregulationofp21andp53in
BMMSCsfromAML,suggestingincreasedcellularsenescence. Importantly,ourresultshighlightthebiologicalandmolecular differencesbetweenAML-MRCanddenovoAMLreportedby otherresearchgroups.13–15
Severallinesofevidenceindicate thatalterationsinthe bonemarrownichecontributetothedevelopmentand pro-gressionofMDSandAMLandoneofthemostcharacterized elementsofthebonemarrownicheistheBMMSCs.9,16 This
cellpopulationrepresentsasmall fractionofbonemarrow nucleatedcells,andastandardizedprotocolforisolationand cultureof BMMSCs may be necessary to minimize experi-mentalvariationsandprovideconclusiveinformationabout thebiologyofthesecellsinthesehematological malignan-cies.Our resultssuggest that upregulation ofCDKN1Aand
TP53mayindicatesenescenceofdenovoAMLBMMSCs,which maycontributetotheineffectivehematopoiesisfoundthethis disease.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Funding forthis work was providedby ConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo
(FAPESP).TheauthorswouldliketothankDr.NicolaConran fortheEnglishreview.
r
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e
r
e
n
c
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MatheusRodriguesLopesa,d,1,
JoãoAgostinhoMachado-Netoa,b,1,FabiolaTrainaa,b,
PauladeMeloCamposa,SaraTeresinhaOlallaSaada,
PatriciaFavaroa,c,∗
aUniversidadeEstadualdeCampinas(Unicamp),Campinas,SP,
Brazil
bUniversidadedeSãoPaulo(USP),RibeirãoPreto,SP,Brazil cUniversidadeFederaldeSãoPaulo(Unifesp),Diadema,SP,Brazil dPresentlyatUniversidadedoValedoSãoFrancisco(UNIVASF),
PauloAfonso,BA,Brazil
∗Corresponding authorat:Universidade Federalde SãoPaulo (Unifesp),DepartamentodeCiênciasBiológicas,RuaSão Nico-lau,210,09913-030Diadema,SP,Brazil.
E-mails:[email protected],[email protected] (P.Favaro).
1Theseauthorcontributedequallytothiswork.
Received14March2016 Accepted4July2016
1516-8484/©2016Associac¸ ˜aoBrasileiradeHematologia, HemoterapiaeTerapiaCelular.PublishedbyElsevierEditora Ltda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.bjhh.2016.07.002
