Gene Expression Profiling during Erythroid Differentiation of K562 Cells

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Mitchell et al. Blood Cells, Molecules, and Diseases (2001)27(1) Jan/Feb: 309–319

doi:10.1006/bcmd.2000.0377, available online at http://www.idealibrary.com on

Gene Expression Profiling during ErythroidDifferentiation of K562 CellsSubmitted 12/02/00(Communicated by H. Ranney, M.D., 12/20/00)

Tim Mitchell,1 Maria Plonczynski,1 Amy McCollum,1 Cheryl L. Hardy,1

Surinder Safaya,1 and Martin H. Steinberg1

ABSTRACT: We studied the temporal changes in gene expression in K562 cells at intervals from 2following induction using differential display polymerase chain reaction and gene expression arraythan 110 cDNA fragments representing 86 unique mRNAs were either up- or downregulated during edifferentiation. Sixty-one of the differentially expressed cDNA fragments had more than 95% homoknown GenBank sequences; 21 represented cDNA sequences with only dbEST or high-throughpscreening database matches. Four fragments had no database matches. Using gene expressiondifferentially expressed genes were observed. Unique expressed sequence tags (ESTs) were usedtwo novel genes from available databases and their tissue expression was examined. Erythroid matinduced K562 cells is associated with differential expression of many genes. Some differentially exclones were transcription factors and 25 expressed fragments with open reading frames were founfunction remains unknown. © 2001 Academic Press

Key Words:gene arrays; differential display PCR; transcription factors; erythroid cells; gene transcription; cDNA expression.

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INTRODUCTION

K562 cells, a human myeloid leukemia cell lican be induced to differentiate by exposure to shchain fatty acids and other polar compounds (1Most induced K562 cell lines expressg-globingenes and fetal hemoglobin (HbF) but some lhave shown a switch fromd- to b-globin genexpression and others can be induced towegakaryocytic differentiation (4). During cell d

erentiation, the pattern of gene expression cha5, 6). Using times of 24 through 72 h postinducte previously characterized some differentiallyressed genes in K562 cells (7). We found m

han 110 differentially expressed mRNA spehat included 25 uncharacterized open rearames (ORFS). Because events happening b4 h after induction may be critical as cells diffntiate we induced K562 cells with sodium buty

Correspondence and reprint requests to: Martin H. Steinberg, DepBoston, MA 02118. Fax: 617-414-1021. E-mail: msteinberg@medic1 G.V. (Sonny) Montgomery Department of Veterans Affairs Medical C

chool of Medicine, Jackson, Mississippi 39216.

309

and studied differential gene expression at interfrom 2 to 24 h. Using differential display polymease chain reaction (ddPCR) and gene exprearrays, we now report a partial profile of gedifferentially expressed during the induction of dferentiation of K562 cells with sodium butyrate. Whave “cloned” two unique genes from public dabases using their differentially displayed ESTsprobes and studied their expression. Some Ohave now been identified, others represent ngenes without known functions, and others monly in the dbEST and high throughput gene scring (htgs) databases. Four mRNAs currentlyany database matches.

MATERIALS AND METHODS

Cell preparation.K562 human erythroleukmia cells were maintained in RPMI 1640 with 10

nt of Medicine, Boston University School of Medicine, 88 E. Newedu.and Division of Hematology, Department of Medicine, University of Mpi

1079-9796/01 $35.00Copyright© 2001 by Academic Press

All rights of reproduction in any form reserved

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Blood Cells, Molecules, and Diseases (2001)27(1) Jan/Feb: 309–319 Mitchell et al.doi:10.1006/bcmd.2000.0377, available online at http://www.idealibrary.com on

bovine calf serum (GIBCO, Grand Island, NLog-phase cells were induced to differentiateincubation with 5 mM sodium butyrate in H2O and

ere harvested after 2, 4, 6, 12, 24, and 48nduction. Globin biosynthesis was verified by muring the activity of a transducedAg-globin gene

promoter as previously described (8).

RNA preparation.Induced and control celwere washed twice in RPMI without serum. ToRNA was isolated and prepared as describedTotal RNA isolated from induced and contcells served as templates for semiquantitaPCR analysis and as substrate for Northern ayses.

To prepare mRNA for Clontech Atlas cDNExpression Arrays, K562 cells were induced w5 mM Na butyrate and control cells were moinduced with diluent. At intervals of 2, 4, and 62 3 107 cells were harvested from induced acontrol culture flasks, centrifuged and the pelsnap frozen in liquid N2 and stored at280°C forRNA preparation. RNA was isolated using Clotech Atlas Pure Total RNA Labeling systewhich is a phenol- based method. Total RNA wDNase treated and the product was assessepurity and yield spectrophotometrically and informaldehyde agarose gel. Total RNA fromduced and control K562 cells was enrichedpoly(A1) RNA and 32P-labeled cDNA probe

ere synthesized from the poly(A) mRNA aurified. Probes prepared using this labeling

em had an activity of 0.5–53 106 cpm and weruitable for hybridization to human cDNA exprion arrays.

Differential display.Differential display PCRas done as previously described (7). PCR pcts were separated by electrophoresis oncrylamide, 8 M urea gels in Tris-borate EDTried gels were exposed to X-ray film and exa

ned for the presence of differentially displayands compared with controls (7).

Selected gel bands were excised fromried gels, eluted in H2O and re-amplified by PC

under the same conditions as the initial reacbut without added35S and purified through
QIAGEN PCR purification spin column. These
310

r

same bands were reexamined during studies okinetics of differential gene expression by exing them from the gel and resequencing to cfirm their identity with the originally identifiebands (7). In kinetic studies we used primer cbinations only for cDNA fragments that mighttranscription factors or, that in our original stuies, lacked homology to known genes.

Sequencing amplified gel bands and databsearches.Part of the amplified gel band was usas a template for cycle sequencing (SequithEpicentre Technologies, Madison, WI), usprimer combinations from the initial displaysequencing primers. Some differentiallypressed mRNA could not be sequenced direand were cloned before sequencing (7).

Confirmation of differential gene expressiWe used both semiquantitative PCR and Northanalysis to verify that gene fragments wereferentially expressed. The Titan One Tube RPCR system (Roche) was employed with scially designed nested primers for semiquantive PCR. Nested primers were synthesized asequencing cDNA fragments isolated from diffential display gels and cDNA was amplifiedrectly from total cellular mRNA at each timpoint studied.

Northern blot analysis.Ten micrograms of total RNA was loaded into each lane of a 1% arose-LE gel (Ambion, Austin, TX) and probed w[32P]dATP-labeled cDNA according to the Ge-

unter Hot Prime method (cDNAs originated froCR amplification of the eluted differential dispands). Hybridization was carried out with stand

echniques. Blots were exposed to X-ray film,eloped and quantified as described (7).

Hybridization. Radioactively labeled probere immediately hybridized to Clontech Humematology Arrays according to a standardridization protocol. Blots were exposed to stard X-ray film at270°C or to a phosphor scre

at room temperature and scanned by a MolecDynamics Personal Densitometer S1 or a STO
optical scanner respectively and quantified using

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ImageQuant software. To achieve normalizaof hybridization signals an average backgrowas chosen from two different areas, one fromarea on the phosphorimager correspondinspots for prokaryotic genes and the other fromarea corresponding to no spotted genes.background was subtracted from the fixed voluof each spotted cDNA. A fold difference andabsolute value difference were determinedeach hybridization signal. Generally, a twofdifference and higher was considered signific

Bioinformatics.Comparative sequence searcere performed using the BLAST algorithm on

ollowing databases: GenBank nonredundant dase, EMBL, DDBJ, and PDB, high-throughene screening (htgs), dbEST, GSS, Interwissprot, and VecScreen (to screen for vector

amination).Curatools software (Curagen) was used

dentify ORFs and the resultant translateduence was entered into the “BLOCKS” databcompilation of multiply aligned unmapped sents corresponding to highly conserved reg

n similar proteins. Sequences which matchepparent coding sections in posted clones wntered into GENSCAN (Stanford University)etect gene promoters, intron- exon junctions9 end-processing signals.

ESULTS

isplay of Differentially Expressed Sequence

K562 cells with an average 62-fold increasg-globin gene expression were used as the soof RNA for differential display. Cells were havested at intervals of 2 to 48 h after their indtion. As previously reported, approximately 1bands appeared to represent differentiallypressed mRNA species; the same cDNA fments were consistently present in multiple gfrom multiple cell inductions. Most differentialexpressed mRNAs were increased comparecontrol cells; some were decreased; a minorepresented mRNA species not present in cocells (see below). Figure 1 shows part of a dif
ential display containing several mRNA species:
311

Fragment D12 which is a newly described g(see below); D22;aNAC transcriptional coactvator; D26 (D15a) with 39 end homology to thyroid receptor interactor mRNA; P27 whichHZF2 mRNA for a zinc finger protein.

Kinetic Analysis of Differential Gene Express

Changes in expression of differentially epressed mRNAs detected by ddPCR are showFigs. 2 and 3. Figure 2 depicts the exprespattern of 32 differentially expressed genes wdatabase matches grouped into 6 arbitrary caries. Half the genes had increased expressionhalf had decreased expression during the sinterval.

Figure 3 presents data from 25 genes thatmatches only in the non-redundant, dbESThtgs databases including four transcripts thatno match in any available database. Seventypercent of these genes had increased expreand 28% showed decreased expression over

FIG. 1. Portion of a ddPCR gel showing differentexpression of cDNA fragments D12, D 22, D26, and P2intervals from 2 to 48 h. Each group of seven lanes resents a control uninduced sample and mRNA harvestereversed transcribed with different anchored primersarbitrary amplimers after 2, 4, 6, 12, 24, and 48 hexposure of K562 cells to 5 mM sodium butyrate.

To verify that the gene fragments we recov-

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ered from differential displays analyzing thenetics of expression were identical to gene fments we isolated in our original studies (7)sequenced these fragments. We were able tsequence 83% of these fragments which widentical to the originally isolated fragments. Senteen percent of fragments were not possibsequence directly.

Figure 4 shows differential gene expressioa human hematology array probed with mRisolated from K562 cells at 2, 4, and 6 h postinduction. The expression of 73 genes, groupedseven functional categories, changed followinduction (Fig. 5). Seventy-three percent of ge

FIG. 2. Expression of genes with GenBank homolasterisk had their expression trend estimated by RT-PClegend shows expression relative to baseline (1:1) usinless than baseline and, 2- to 4-fold, 5- to 10-fold, and

had increased expression during the study interval

312

and 27% had decreased expression. Twelve gdifferentially expressed in the array analysis wtranscription factors.

Northern Blot and Semiquantitative PCRAnalysis of Differential Expression

Thirteen cDNA fragments whose differentgene expression was confirmed by Northernanalysis are shown in Fig. 6. An additional secDNA fragments whose differential expresswas verified using semiquantitative PCRshown in Fig. 7. The identity of these fragme

during time intervals of 2 to 48 h. Some genes shownit was not possible to do this accurately on the ddPCR

ayscale depicting 2- to 4-fold less than baseline, morethan 10-fold above baseline.

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Cloning Partially Characterized ESTs

Figure 8 shows the sequence of two cDfragments, D12 and P30, isolated from a difential display and Fig. 9 shows the expressio

FIG. 3. Expression of 25 genes with matches only inhat had no match in any available database. Legend isstimated by RT-PCR.

FIG. 4. Human hematology gene-related cDNA exand 6 h following induction with Na butyrate. The uppe
test sample (induced).
313

these genes by ddPCR, Northern analysisRT-PCR. Some properties of these genesshown in Table 1.

The expression of D12 and P30 in differtissues is shown in Fig. 10. D12, characterized

on-redundant, dbEST and htgs databases including 4ame as for Fig. 2 and asterisks mark genes whose exp

ion array probed with mRNA isolated from K562 cellsl of each set is a control (uninduced) and the lower pa

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mRNA of approximately 1.8 kb, appears toexpressed primarily in brain. P30, with mRNAsapproximately 2.6 and 4.0 kb is expressedheart, skeletal muscle, kidney and placenta. Mtiple mRNA bands may represent alternamRNA splicing, alternative transcription stsites or other mRNA processing differences.sequence of P30 has been recently depositeGenBank as Accession No. AF255443 as a n

FIG. 5. Differential expression of 73 genes at 2, 4, ad 6array. Shown are fold increases or decreases from bagrayscale depicting 2- to 4-fold less than baseline, moa more than 50-fold above baseline.

human gene by comparative proteomics.

314

DISCUSSION

High-throughput screening methods suchdifferential display PCR, filters containing cDNfrom hundreds of genes, and microarrays ofof thousands of genes have permitted the profiof gene expression in quiescent and differening cells, normal and neoplastic cells, and cunder experimental or pathological conditions

stinduction detected using a human hematology-related. The legend shows expression relative to baseline (1n 5-fold less than baseline and, 2- to 10-fold, 1-1 to 50

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cell may be detected. Computational sciencedevised mathematical methods for clusteringpression patterns. Soon, microarrays with thetire genome will be available. We used commentary methods of ddPCR and gene expresarrays to study the profile of gene expressioK562 cells, a human erythroid cell line. UsiddPCR, genes that are already known or mRwith homology to known genes can be detecand unique ORFS characterized. Gene exprearray technology may be useful in exploitingdifferential expression of known “candidagenes and genes of unknown function; instudies an expression array containing humanmatology-related mRNAs was selected.

Gene expression has been studied in erypoietin responsive CD71-positive cells isolafrom human blood using a two-phase liquid cture system (6). Subtractive hybridization w

FIG.

used to identify genes that were up-regulated in r

315

proliferating cells. Expressed genes includedthat were already characterized, 170 uncharaized genes that had matches in databases anovel transcripts. Among the characterized ge29% were involved in transcription and replition and 22% in translation. Globin transcrirepresented about 1% of sequenced clones.

Quiescent, serum deprived human fibroblaalmost immediately begin to express transcripfactor genes and genes involved in signal trduction when exposed to serum (5). Some of thgenes were expressed only transiently whileers had more sustained expression after inducOther genes expressed in quiescent cellsdown-regulated following induction. Genes wlater expression, like the HLH proteins Id2 aId3—genes we found to be expressed in Kcells and others found to play a possible roleexpression of theg-globin genes—may act

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these studies using microarrays containing mnearly 9000 cDNAs, more than 200 partially chacterized ESTs whose function was unknownspecific patterns of temporal expression.

FIG. 6. Northern blot analysis of cDNA fragmenamplified from ddPCR gels. Two groups of blots (A andare separated because of the use of different loadingtrols of 18S RNA. Fragments C11b, P23, and P7 werein the GenBank nonredundant database at the timearticle was prepared.

FIG. 7. Analysis by semiquantitative RT-PCR of cD5 mM sodium butyrate. mRNA was isolated at intervalwas amplified directly from total cellular mRNA. Percen
integrated density analysis (Stratagene).
316

Our goal was the discovery oftrans-actingactors that may activateg-globin gene expreion. K562 cells, an erythroid cell line that eresses theg-globin gene when induced to diffentiate by incubation with the short-chain facid, sodium butyrate, was used as a moderythroid differentiation. This agent may be u

ul clinically and can increase HbF concentratin patients with sickle cell anemia (14). IncreasbF levels in sickle cell anemia is clinically beficial (G315). Induction of globin gene exprion was confirmed by measuring the activity oransduced Ag-globin gene promoter. Manevents occur during K562 cell differentiation athe expression of a transducedg-globin gene promoter strongly suggests that the transcriptioenvironment needed forg-globin gene expressios present (16). Initially we found more than 1ifferentially displayed cDNA fragments that aeared to originate from either up- or downre

ated genes (7). Semiquantitative PCR and Norn blot analysis, isolating mRNA directly fro

nduced cells at different times after inductonfirmed that gene fragments were differentixpressed. Among the 110 differentiallyressed cDNA fragments, 86 unique sequeere found. Fifty-four fragments represented p

eins whose structure was known and whose fion had been at least partially described. Se

fragments isolated from K562 cell induced to differenti2 to 24 h and the cDNA fragment, first identified by

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fragments were homologous to sequences indatabases but lacked structural or functional sies (June 2000).

Eleven fragments isolated during the studof expression from 2 to 48 h had dbEST matcand 10 fragments had matches in the htgsbase. Most of these fragments have been assto chromosomes 1, 9, 10, 12, 13, and 16. Ffragments, D2b, D9, D24, and P41, had no dbase match. Of the fragments that repre

FIG. 8. Sequen

FIG. 9. Expression of gene fragments D12 and P3ddPCR, Northern blot analysis, and semiquantitativePCR. Northern blots contained 8mg of total cellular RNAper lane and RT-PCRs contained 200 ng of cellular RFor controls, 18S RNA was used for the Northern ana
and GAPDH and c-myc served as controls for RT-PCR.
317

known proteins we cloned transcription factthat included: Differentiation-related gene-1 (d1); PAX 3/forkhead transcription factor; HZF2Kruppel-related zinc finger protein; heir-1, Idand GOS8, helix–loop–helix proteins;aNACranscriptional coactivator; LIM domain proterophoblast hypoxia regulated factor (7). Theential importance of some of these transcripactors is suggested by the studies of Holmeso-workers who used a fungus-derived compoOSI-2040) which inducesg-globin gene expre

sion to probe forg-globin regulatory genes thre differentially expressed in induced and uuced cells K562 cells using representational

erence analysis (17). In this analysis, expresf the gene for the helix-loop-helix (HLH) tracription factor Id2 was increased coordinaith g-globin. Sodium butyrate also up regula

the Id2 gene and overexpression of Id2 in Kcells reproduced the enhancement of endoge

f genes D12 and P30.

g-globin expression. An E-box element in hyper-

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sensitive site 2 of theb-globin gene cluster locuontrol region was required for Id2-dependnhancement ofg-globin gene promoter activi

suggesting that E-box site occupancy by bHLH proteins may influence this activity. We adetected increased expression of the Id2 genhematology expression arrays.

We used two differentially expressed ESD12 and P30, to “clone” from existent data batwo previously undescribed genes. The sequof these putative genes is unrelated to kngenes and their expression is not restricteerythroid cells. D12 appears to be expressebrain and its putative protein has homology

TA

D1

Chromosome location 1Size in bp (Northern analysis) ;1800Expression pattern Increases to 24 hHomology

Interpro TropomyosinblastX Low homology to man

BLOCKS Tyrosine-specific proteGenescan size (bp) 1788, 1800, 2286Exons matched by cDNA sequence 4Total exons in gene 13 (for 1800-bp geneEnsembl gene YesTissue distribution 1.8-kb mRNA, brain,

intestine

FIG. 10. Northern blot analysis of tissue expression(Clontech) were 325- and 300-bp cDNA fragments ePrime). Lanes: 1, brain; 2, heart; 3, skeletal muscle; 4,
placenta; 11, lung; 12, blood leukocyte.
318

tropomyosin. P30 is widely expressed in at leatissues and has homology to TPR (tetratricoptide) repeats, a repeat of 34 amino acids impoin protein-protein interactions (18).

As expected, erythroid maturation in K5cells is associated with differential expressover time of numerous genes. By studyingprofile of gene expression as K562 cells begiexpress theirg-globin genes, identifying new

xpressed transcripts that play key roles inctivation of globin gene expression and in es

ishing the propertrans-acting environment foigh levelg-globin gene expression may be pible.

1

P30

20;2600

Decreases during 24 h

TPR repeatDrosophila cell-cycle control protein,crooked neck

sphatase No matches2784216

No.0-kb band in small 2.6- and 4.0-kb mRNA, primarily in hea

placenta, skeletal muscle, kidney

gments D12 and P30. Probes for the 12-lane human tisfrom ddPCR gels labeled with [a-32P]dATP (GenHunter Ho; 5, thymus; 6, spleen; 7, kidney; 8, liver; 9, small inte

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ACKNOWLEDGMENT

This work was supported by Research Fund ofepartment of Veterans Affairs (Initiative for Histoally Black Colleges and Universities).

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Gene Expression Profiling during Erythroid Differentiation of K562 Cells

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