wilms’tumorgene, wt1, mrna isdownregulated...

Vol. 5, 677-686, june 1994 Cell Growth & Differentiation 677

Wilms’ Tumor Gene, WT1, mRNA Is Downregulatedduring Induction of Erythroid and MegakaryocyticDifferentiation of K562 Cells

Shelley A. Phelan, Charles Lindberg, andKatherine M. Call2

Department of Molecular and Cellular Toxicology, Harvard School of

Public Health, Boston, Massachusetts 021 15

Abstrad

Evidence implicates the produd of the Wilms’ tumorsuppressor gene, WT1, in proliferation anddifferentiation of target tissues during development.Study of the regulation of other tumor suppressor genesduring these processes has been instrumental in definingtheir interadions and fundions. In this study, weperformed experiments to assess the suitability of thehuman K562 erythroleukemia cell line for studying theWT1 gene during differentiation. We predided thatWT1 mRNA would be decreased during indudion ofdifferentiation of K562 cells based on observations ofdecreased WT1 mRNA during kidney development andin differentiated Wilms’ tumors and leukemias.Accordingly, we found that WT1 mRNA was down-regulated in K562 cells during indudion of erythroidand megakaryocytic differentiation by sodium butyrateand 1 2-O-tetradecanoylphorbol-1 3-acetate, respedively.Down-regulation of WT1 mRNA was not a generalizedphenomenon of growth inhibition. WT1 mRNA was notdown-regulated when 1 2-O.tetradecanoylphorbol-1 3..acetate-induced differentiation was blocked bybryostatin-1 . During 1 2-O-tetradecanoylphorbol-1 3-acetate treatment, the decrease in WT1 mRNA wasrapid (within 5 mm), continuous, and occurred, at leastin part, posttranscriptionally. An analysis of the 5’flanking region and transcription initiation sites of thehuman WT1 gene also was performed. Our data suggestthat K562 cells will be a valuable system for unravelingsignal transdudion pathways by which WT1 is regulatedand for investigating the interadions and role of WT1 indifferentiation.

Introdudion

W-r,3 or nephroblastoma, is a kidney tumor which occurs ata frequency of 1 in 1 0,000, predominantly in infants and

Received 1/25/94; revised 4/4/94; accepted 4/8/94.

1 This investigation was supported in part by a Milton Fund Award (to

K. M. C.), Center Grant ES00002 from the NIEHS, and an award from theLucille P. Markey Charitable Trust to the Department of Molecular andCellular Toxicology. S. A. P. was supported by PHS Training Grant 5 T32-CA-09361 -1 2 and institutional funding provided by the Division of MedicalSciences at Harvard Medical School.2 To whom requests for reprints should be addressed, at Department ofMolecular and Cellular Toxicology, 1 -1 05, Harvard School of Public Health,665 Huntington Ave., Boston, MA 02115.3 The abbreviations used are: WT, Wilms’ tumor; cDNA, complementary

DNA; IGF, insulin-like growth factor; IGFIR, IGF 1 receptor; PDGFA, plate-let-derived growth factor A chain; PDGFB, PDGF f3 chain; TPA, 12-0-tetradecanoylphorbol-1 3-acetate; TGF, transforming growth factor; DMSO,

young children (1). Based on positional cloning strategies,an 1 1 p1 3 Wilms’ tumor suppressor gene, WT1, has beenisolated (2, 3). The encoded polypeptide possesses classic

cys-his zinc finger motifs and an amino terminal composi-(ion similar to the EGR-1 and EGR-2 proteins (2, 3). The M�52,000-54,000 WT1 protein (4) has been demonstrated tobe a transcription factor (5). Two alternative splices whichresult in four WT1 mRNA species have been identified (6).Mutations of the WT1 gene have been observed in WTs andseveral other tumors (reviewed in Ref. 7).

Multiple lines of evidence suggest that during develop-ment, the WT1 product exerts a role in the proliferation anddifferentiation of renal, urogenital, and other cells in whichit is expressed. WT1 mRNA is expressed primarily in thecondensing mesenchyme, renal vesicles, and the podocyteface of the glomerulus during fetal kidney development (8).Clinical evidence indicates that WT arises from early kidneymetanephric blastemal cells which have failed to differen-tiate properly (9). The findings that the WT1 protein is atranscription factor which is mutated in a subset of Wilms’tumors suggest that this protein exerts a pivotal role incontrol of kidney cell growth and/or differentiation pro-cesses. Expression of the WT1 gene in the renal and uro-genital system during embryogenesis (8, 10) and the pres-ence of renal and urogenital anomalies in humans (1 1 , 12)and mice (1 3) with intragenic mutations of the WTJ genedemonstrated a developmental function for WTJ in thesetissues. A role for WT1 protein in hematopoietic cells hasbeen suggested based on its expression in leukemia celllines, spleen tissue, and a pre-B-cell cDNA library (2) andan inverse correlation between the expression of WTJ

mRNA and differentiation status of human leukemia cells(14, 15).

The mechanisms of action and regulatory targets of WTJ

protein are beginning to emerge. WT1 protein has beenreported to bind the EGR1 binding site (1 6). The presence ofthe alternatively encoded three amino acids (KTS) betweenthe third and fourth zinc fingers of WT1 alters the DNAbinding specificity (1 7). The finding that WT1 protein (-KTSforms) can bind to the EGR1 binding site, along with keyobservations about other growth-related genes, led to theidentification of genes potentially regulated by WTJ pro-tein, which are in accord with the predicted roles of WTJ

protein. IGF-lI (18), PDGFA (19) and insulin-like growthfactor 1 receptor (IGFIR) (20) promoter sequences havebeen demonstrated to be transcniptionally repressed byw-ri protein in transient expression assays. There also isevidence that the WT1 protein can act as a transcriptionalactivator (21, 22).

Unlike Rb, the choice of cell lines in which to delineatethe regulation, interactions, and functions of endogenous

dimethyl sulfoxide; gpllla, glycoprotein lIla; HS, horse serum; bryo, bryosta-tin-i ; bp, base pair(s); TRE, TPA responsive element; PBS, phosphate-buffered saline; ORF, open reading frame.

to0

x

.0EC

0

A

B

10

.f

0

678 Down-regulation of WT1 mRNA during Differentiation

4 A. Memisoglu and K. Call, unpublished results.

w-ri during differentiation (and proliferation) is greatly lim-ited by the relatively few cell lines demonstrated to expressthis gene. To date, there is no human kidney cell linereported to express WT1 which has been characterized andused for renal differentiation studies. We have utilized theK562 cell line since it is of hematopoietic origin, expressesw-ri mRNA (2) and protein (23),� and can be induced todifferentiate along multiple lineages with a variety of chem-icals (reviewed in Refs. 24 and 25). The K562 cell line wasestablished from a patient with chronic myelogenous leu-kemia in blast crisis and possesses a Philadelphia chromo-some (26). The K562 cell line is negative for expression ofB- and T-cell antigens and exhibits relatively undifferenti-ated erythroid characteristics (27-29).

In this study, we examined the expression of the WT1gene in K562 cells during differentiation to gain insightsinto its regulation and interactions. We also performedthese investigations to evaluate the suitability of the K562cell line for studying the role of WT1 in differentiation. Asdiscussed previously, the inverse correlation between WT1mRNA levels and differentiation status during kidney de-velopment (8, 1 0), ofWilms’ tumors (30-32) and leukemias(1 4, 1 5), and the failure of metanephnic blastemal cells todifferentiate in WTJ knock-out mice (1 3) strongly indicatesa role of the w-ri protein in differentiation. However, therehas been no demonstration of a cell culture system suitablefor studying the WT1 gene during differentiation. The iden-tification of a cell culture model would be important inunraveling the mechanisms of WT1 regulation, key regula-tory interactions, and signal transduction pathways, all ofwhich are essential for understanding the functions of WT1protein during differentiation. One of the first criteria for anappropriate system would be the down-modulation of WT1during differentiation. Accordingly, the investigationsherein showed that WT1 mRNA was down-regulated dun-ing differentiation of K562 cells towards both the erythnoidand megakaryocytic lineages. Given these results, we ex-amined the kinetics and mechanisms of regulation of WT1mRNA during differentiation and potential relationships toother events implicated in the differentiation of K562 cells.We sequenced the human WTJ 5’ flanking region andmapped transcription start sites in the K562 and CEM hu-man leukemia cell lines to gain additional informationabout the regulation of Wfl.

Results

Down-Regulation of the Steady-state WT1 mRNA Levelduring Differentiation of K562 Cells along Erythroid andMegakaryocytic Lineages. To determine whether WT1 isdown-regulated during K562 differentiation, we examinedthe effect of two chemical inducers on the steady-state levelof WTJ mRNA. We used sodium butyrate, which inducesmultiple properties of erythroid differentiation (28, 33, 34).We also used TPA, which induces a megakaryocytic phe-notype in the K562 cell line based on antigen expressionpatterns (35). During TPA-induced differentiation, trans-forming growth factor beta 1 (TGFf31) (24) and erythroidpotentiating activity/tissue inhibitor of metalloproteinasemRNAs are increased (36) and expression of PDGFB (c-sis)and c-fos mRNAs are induced (37, 38, 39). In our expeni-ments, cultures of K562 cells in logarithmic (log) growth

1 2 3 4

Time in days

1!�

wri�>R�.1

TGFf�1-)�

PDGF�-* #{149}�

�n->

Fig. 1. Analysis of K562 cells during induction oferythroid and megakaryo-

cytic differentiation. A, K562 cell growth during sodium butyrate and TPAtreatment. K562 cells were mock (#{149}),0.1% DMSO (V), 1 msi sodiumbutyrate fY), and 3 nM TPA (0) treated. Growth was monitored daily andplotted as described in “Materials and Methods.” B, Northern blot analysisduring sodium butyrate and TPA treatment. K562 cells were treated asdescribed in A. Total RNA was isolated on day 2, Northern blotted, andhybridized with madiolabeled probes for WT1, TGF(31 , PDGFB (c-sis), and�-actin (control) cDNAs.

phase were mock, DMSO (0.1 %), sodium butyrate (1 mM),

and TPA (3 nM in 0.1 % DMSO) treated for 2-4 days asdescribed by Alitalo et a!. (38). As expected, both TPA andsodium butyrate treatment inhibited cell growth markedlycompared to mock and DMSO controls (Fig. 1A).

We analyzed the relative steady-state level of WT1mRNA and parameters of differentiation in sodium butyrateand TPA-treated K562 cells. Northern blot analysis showedthat WT1 expression was decreased markedly in both so-dium butyrate and TPA-treated cultures compared to mockand DMSO controls on day 2 (Fig. 1 8) and day 4 (data notshown). There was, on average, a 21 - and 8.3-fold decreasein W11 mRNA in sodium butyrate and TPA-treated cul-tunes, respectively, on day 2 and comparable decreases onday 4. Treatment of K562 cells with 1 mt�’i sodium butyrateresulted in a progressive increase in expression of fetalhemoglobins, demonstrating induction of erythroid differ-entiation. We typically observed 30-35% benzidine-posi-tive cells in sodium-treated cells versus 1 % in controls,similar to published observations (34, 40). K562 cellstreated with TPA for 2 days showed a 5- to 21 -fold induc-(ion of gpllla, a specific marker of megakaryocytic differen-

to0

.0EC

A

B

I 2 3

Time in days

Cell Growth & Differentiation 679

5 B. Dooreck and K. Call, unpublished results.6 5 Phelan and K. Call, unpublished results.

tiation. Together, these data demonstrated the down-regu-lation of WT1 mRNA during induction of erythroid andmegakaryocytic differentiation.

We also examined the mRNA levels of two other genes,TGFf31 and PDGFB, implicated in growth and differentia-(ion whose expression has been reported to be up-regulatedduring megakaryocytic differentiation of K562 cells. Weanticipated that we could gain insights into potential regu-latory interactions by comparing the expression of theseand other genes implicated in growth and differentiation ofK562 cells with WT1 mRNA levels. In cells treated with TPAfor 2 days, we found a 7-fold increase in TGFf31 mRNA andan induction of PDGFB mRNA (Fig. 1 B). Alitalo et a/. (24)have reported similar increases during TPA induction ofmegakaryocytic differentiation of K562 cells.

Decrease in the Steady-State WT1 mRNA Level Is NotAttributed to Inhibition of Cell Growth. Given that bothinducers of differentiation inhibited proliferation (Fig. 1A)and the potential involvement of WT1 in proliferation, itwas formally possible that the down-regulation of WTJmRNA could be due to cessation ofcell growth. This did notappear likely since TPA caused a greater inhibition of cellgrowth than sodium butyrate (Fig. 1A), yet did not result inas great a decrease in the level of WT1 mRNA (Fig. 1 B).

However, to investigate this possibility, we examined theeffects of serum starvation and other treatments which in-hibit cell growth on the steady-state level of WT1 mRNA. Inthe serum-starvation experiments, asynchronous K562 cellsgrown in RPMI 1640 supplemented with 10% heat-macti-vated HS were pelleted, resuspended in media plus 0.5% or1 0% HS, and monitored daily for 72 h. Fig. 2A shows thatserum-starvation resulted in growth inhibition comparableto that observed during sodium butyrate treatment (Fig. 1A).Northern blot analysis, however, showed that the relativelevel of WT1 mRNA was comparable (less than 1 .5-foldvariation) in serum-starved (24 and 48 h) and asynchronousK562 cells (Fig. 2B). Although the mechanisms of thisgrowth inhibition are likely different than in response tosodium butyrate or TPA, the serum starvation experiments,nonetheless, showed that down-regulation of WT1 mRNAwas not a generalized phenomenon of growth inhibition. Inaddition, we found that several other chemicals and gammairradiation treatment, which inhibit cell growth, had noeffect on the steady-state level of WT1 mRNA.5

Within the serum-deprivation experiments, we also as-sessed whether the WT1 gene could be classified as animmediate-early growth response gene in the K562 andseveral other cell lines. We undertook these investigationssince the WT1 product has similarities to the EGR-1 andEGR-2 proteins, and the WT1 5’ flanking region possessesserum response-like elements (to be discussed). We ob-served a marginal (2-fold) but reproducible increase in WT1mRNA in K562 cells 30 mm after serum-restimulation(Fig. 2B). There was an induction in the level of EGR1mRNA, demonstrating that serum-stimulation was effectiveat inducing an immediate-early growth response. The lackof a marked increase in WT1 mRNA in serum-stimulatedK562, as well as NIH3T3, BSC-1 , and CEM cell lines,6indicated that WT1 cannot be considered a classic imme-diate-early growth response gene.

.C .C� �

E #{176}��2 c6u��II

g � �i-000

WTI -> �

egr-1 -> � � �4 0

�-acUn -3 �

Fig. 2. Analysis of K562 cells during asynchronous growth, serum starva-tion, and serum induction. A, K562 cell growth during serum starvation.K562 cells were cultured in RPMI 1640 supplemented with 1 0% (#{149})and0.5% (V) heat-inactivated HS (24 and 48 hI, and growth was monitored dailyand plotted. B, Northern blot analysis. K562 cells were treated as describedin A. In addition, an aliquot of cells grown in medium plus 0.5% HS (48 hiwas resupplemented (stimulated) to a final concentration of 10% HS for 30mm. Total RNA from these cultures was isolated, Northern blotted, andhybridized with w�ri, egr-1 , and �-actin cDNA probes.

Inhibition of TPA-induced Differentiation by Bryosta-tin-i Blocks the Down-Regulation of WT1 mRNA. Ifdown-regulation of WT1 was involved in differentiation,then blockage of induction of differentiation should inhibitthe decrease in the steady-state level of WT1 mRNA. InK562 cells, bryo is capable of inhibiting megakaryocyticdifferentiation induced by TPA (41). Bryo is a protein kinaseC (PKC) activator but is structurally unrelated to TPA andactivates a different subset of protein kinase C isotypes inK562 cells (41). Simultaneous treatment with 100 nM bryoeffectively inhibits the induction of megakaryocytic differ-entiation by TPA in K562 cells, but treatment with 1 nM bryodoes not (41 ). To our knowledge, there are no reportedconditions for inhibition of sodium butyrate-inducederythroid differentiation of K562 cells.

We evaluated the effects of TPA and bryo on cell growth,wri expression, and differentiation status. Specifically, wewanted to know whether the inhibition of TPA-induceddifferentiation by bryo prevented the down-regulation ofw-ri mRNA. The data in Fig. 3A showed that simultaneoustreatment with 1 00 nM bryo, but not 1 nM bryo, was effec-tive at alleviating the growth inhibitory effect of TPA. North-em blot analysis of this experiment (Fig. 3B) showed thattreatment with bryo (1 or 100 nM; Fig. 3B, Lanes 3 and 4,respectively) alone had no significant effect on the level ofw-rl mRNA, compared to untreated (Fig. 3B, Lane 1) andDMSO treated (Fig. 3B, Lane 2) controls. Cultures treated

A

0

B

WT1

Time in daysB

�. �

3.0

2.5

2.0

1.5

1.0

0.5

0

simultaneously with 3 nM TPA plus 1 nM bryo (Fig. 3B,

Lane 5) and 3 nM TPA alone (Fig. 3B, Lane 7) eachshowed a 1 0-fold decrease in the level of WTJ mRNA.However, cultures treated with TPA plus 100 nM bryo(Fig. 3B, Lane 6) exhibited only a 2.5-fold decrease inw-ri mRNA. Induction of gpllla by TPA was not inhibitedwhen cells were treated simultaneously with 1 nM bryobut was inhibited substantially by 1 00 nt�’i bryo (data notshown), as has been reported previously (41). Thus, theresults of the TPA and bryo experiments (Fig. 3B anddata not shown) demonstrated a concordance betweenthe down-regulation of WTJ mRNA and induction ofmegakaryocytic differentiation.

Time-dependent Analyses during TPA Indudion ofMegakaryocytic Differentiation. We performed a timecourse analysis of the steady-state levels of WTJ, TGF/31,and PDGFB transcripts during induction of differentiationby TPA. This was done to gain insights into the mechanismsand kinetics of WT1 mRNA regulation during differentia-tion, possible regulatory interactions, and the functionalsignificance of the down-regulation of WTJ mRNA. North-em blots (Fig. 4 and data not shown) showed that thedecrease in WT1 mRNA declined to 50% by 5 mm afterTPA treatment (Fig. 4, A and B). A maximal reduction (7- to14-fold) in WT1 mRNA was reached by 4 h and persisted at24 (Fig. 4; A and B), 48 (Fig. 1 8), and 96 h (data not shown),hence during and after the induction of differentiation.The down-regulation of WTJ mRNA by TPA occurred in

the presence of cyclohexamide, indicating that proteinsynthesis is not required (data not shown).

We compared the steady-state levels of WT1, TGF�1,and PDGFB transcripts to assess whether there were anyindications of negative transcriptional regulation of theTGFg31 or PDGFB genes by Wri. This was investigatedbecause: (a) WT1 protein is capable of transcniptionallyregulating several growth factors (1 8-20); (b) the TGFf31(42) and PDGFB (43) promoters have potential EGR1 bind-


.(a.

0 �O �‘� �� I-

� �

Fig. 3. Analysis of K562 cells during treatment with TPA and bryostatin. A,K562 cell growth. K562 cells were mock (0), 0.1 % DM50 (#{149}),and 3 nsi TPA(L\), 1 00 nM bryo fY), 1 nsi bryo (V), 3 nta TPA plus 1 00 nsi bryo (U), and 3

nM TPA plus 1 nM bryo (0) treated. Growth was monitored daily and plotted.B, Northern blot analysis. Cultures of 1(562 cells were treated with TPA andbryo for 2 days as described in A. RNA was isolated from these cultures,Northern blotted, and hybridized with WT1 and 36B4 (control) cDNAprobes.

a)>a)

z

Ea)>

0)

A WA� cc

5 �o �

0 so �- �5 �- c,a �

wni -4 � �

TGF�1 -* �::::-�- 11111

PDGFB -* - �

3684-)�

0 1 2 3 4 24

Time in hours

WG F

Fig. 4. Time-dependent analyses of TPA-treated K562 cells during induc-tion of megakaryocytic differentiation. A, K562 cells were mock (control) or3 nsa TPA-treated for intervals between 5 mm and 24 h. Total RNA wasisolated from untreated and TPA treated cultures, Northern blotted, andhybridized with radiolabeled WT1, TGF�1 , PDGFB, and 36B4 (control)cDNA probes. Northern blots from this and a duplicate experiment wereexposed in the linear range and analyzed densitometrically as described in“Materials and Methods.” B, data from these experiments were graphed asthe relative levels of WT1 (#{149})and TGF�31 (0) mRNAs versus time. Therelative levels of PDGFB mRNA were not quantified due to low expressionin untreated controls. C, induction of gpllla expression during TPA treatment.K562 cells in log phase were treated with 3 nM TPA, and the relativeexpression of gpIIla was determined at 0 (-), 6 (- - - - ), 24 ( . .), and 48(. . .) by FACS analysis as described in “Materials and Methods.” TheFACS data was plotted as the logarithm of relative fluorescence intensity(log F) versus the number of cells (n).

ActD

�-. L� I�

_C ..C .C .C

1-c�� CO

w


-� CC� C.---

0 LL)OOLn,�..c1)

WT1 -> �

�-actin -> � �

Fig. 5. Time-dependent Northern blot analysis of Actinomycin D-treated

K562 cells. Northern blot analysis was performed using polyadenylatedmRNA isolated from cells treated with Actinomycin D (5 pg/mI) for intervals

between 5 mm and 6 h. Northern blots were hybridized with WT1 andf3-actin cDNA probes. Northern blots from this and a duplicate experimentwere exposed in the linear range and analyzed densitometrically as de-scribed in “Materials and Methods.”

ing sites; and (C) TGFJ31 and PDGFB have been implicatedin megakaryocytic differentiation of K562 cells (24, 38, 39).During TPA treatment, TGFf31 mRNA levels increased ap-proximately 1 .5-, 2-, and 7-fold by 2, 24 (Fig. 4A), and 48h (Fig. 1 8), respectively. Fig. 4B shows that the increases inthe TGF31 mRNA levels are related inversely to the de-creases in w-ri mRNA levels. However, the increases inTGF�31 mRNA occur shortly after the decreases in the WTJmRNA and continue after the maximal decrease in the WTJmRNA levels (4 h) is reached. The increase in PDGFBmRNA is detected by 4 h and is further elevated at 24 and48 h (Fig. 1 B, Fig. 4B). The fold increases in PDGFB mRNAlevels are difficult to quantify given the relatively low ex-pression levels in untreated cells. Ahitalo et a!. (24) havereported that purified TGF/31 is not responsible for theincreases in PDGFB and TGFf31 mRNAs in TPA-treatedK562 cells. Thus, the data are compatible with WT1 actingas a transcriptional repressor of the TGF31 and PDGFBgenes.

A time-dependent comparison of the alterations in WT1,TGF�1 , and PDGFB mRNA levels with the induction of amegakaryocytic phenotype was made. Fig. 4C shows thatthere is no induction of gpllla expression 6 h after TPAtreatment is initiated. Induction of gpllla is observed at 24 h,

but the mean value is 72% of that observed in TPA-treatedK562 cultures at 48 h. A time course comparison of WTJmRNA (Fig. 4, A and B) and gpllla expression (Fig. 4C)levels showed that the down-regulation of WT1 mRNA ismaximal prior to induction of a megakamyocytic phenotype.For TGF�1 and PDGFB mRNAs, the increases begin prior tothe appearance of a differentiated phenotype and continuethroughout induction.

Analysis of the Half-life of WT1 mRNA. The half-life ofWTJ mRNA was assessed by Northern blot analysis usingpolyadenylated mRNA isolated from K562 cells treatedwith actinomycin D (5 �iWml) for intervals between 5 mmand 6 h (Fig. 5). Densitometnic analysis of the actinomycinexperiments showed that the half-life of WTJ mRNA is 1 .5to 2 h in K562 cells. /3-actin mRNA, which was used as acontrol, exhibited a slight increase in actinomycin-treatedcells with a half-life greater than 12 h, in agreement with

previous reports. The down-regulation of WT1 mRNA inresponse to TPA (Fig. 4A) is more rapid than can be ac-counted for by the half-life (Fig. 5). This indicated that thedecrease in steady-state WTJ mRNA occurred, at leastpartially, at the posttranscniptional level.

DNA Sequence Analysis of the Human WT1 5’ FlankingRegion and Determination of Transcription Initiation Sites.We undertook DNA sequencing of the human WTJgenomic 5’ flanking region (Fig. 6A) to gain insights into theregulation of this gene. In conjunction, we performed Si

nuclease analysis to define the WT1 transcription initiationsites in the K562 and CEM human leukemia cell lines(Fig. 6B). Analysis of the 5’ flanking WTJ sequence me-

S vealed a number of potential regulatory elements. PotentialSpi binding sites (GC boxes; GGCGGG) are located at-628 to -633, +140 to +145, +392 to +397, +635 to+640, and +709 to +714 bp. A tight cluster of sequenceswith marked similarities to the EGR1 (WTJ) binding site(GCGGGGGCG) are found at positions +388 to + 396,+394 to +402, +400 to +408, +410 to +418, and +417to +425 bp. Two other sequences, which conform to theEGR1 binding site in 7 of 9 positions, are located at +624to +632 and +705 to +71 3 bp. The presence of theseEGR1 sites suggests transcriptional autoregulation of theWT1 gene. Of relevance to our differentiation results, a

sequence conforming to a TRE and AP-1 binding site(TGAGACGAG) is found at +263 to +271 bp. This exactsequence in the TGF�3i promoter is required for maximaltranscriptional induction in response to TPA (44). However,if this is a functional TRE for WTJ, our results indicate this

site would confer repression. Theme are sequences withsimilarities to the serum response element immediately up-stream of the + 1 initiation site at positions -6 to -1 5 and-47 to -56 bp, as well as other potential sequences furtherupstream. However, none of these sequences conform ex-actly to the serum response element cone (CC-A/T(,-GG)and flanking consensus sequences and, as presented previ-ously, WT1 does not behave like an immediate-earlygrowth response gene.

Si nuclease mapping revealed three WT1 transcriptioninitiation sites in the K562 cell line and four sites in the CEMcell line (Fig. 6B). The high GC content of the human 5’flanking region posed difficulties for primer extension anal-ysis, as was the case for the mouse sequence (10). In theK562 cell line, transcription initiation sites were observed at+1, +71, and +200 bp. Three identical transcription start

sites were observed in the CEM cell line; however, a fourthstart site also was observed at +556 bp in the CEM cell line,which was not observed in the K562 cell line. The first threesites are too close to be distinguished on Northern blots.While the fourth site should be distinguished by size in CEMcells, its relatively low abundance may preclude its detec-tion. The frequency of use also differed between cell lines(Fig. 68). In the CEM cell line, the +1 transcription initia-tion site predominated, and in the K562 cell line, the majorinitiation site was at +200 bp. As was the case in themouse, no TATAA on CCAAT box sequences were locatedin the vicinity of the transcription initiation sites.

Discussion

We have shown that WTJ mRNA is down-regulated aftertreatment with chemicals which induce parameters of ery-thnoid and megakamyocytic differentiation in K562 cells.These decreases do not appear to be due to inhibition of cell

B

IV

Ill -

II -�

k

I

CA GTGTCTTGTA GAATCTTCAG TGTCTTGATA

ATAATTTTAA AAGCTTCTGA GTGGAGACGA CGCAAAGTCA AGCAGCAAAG GTGGCCTGGG

AGGCAAGCGG AGGGCTCAAG TGCCGCATCT TTACCCTCAG GGTCTCCTGC GCCTACGGGA

TGCGCATTCC CAAGAAGTGC GCCCTTCGAG TAAGTCCTGG GCCCGCACAC ACTTCGGGTC

CGCAGCCAGA ATTTAATGGC GACAATCGTT TATGCAATGC AAGCTAAAAA CCAAAGCGTA

AAAAATTACT ATGTCATTTA TTGAAACGCC ATTCTTTGTC AAACTGCAAC TACTTTGCTT

CACATAAGTT TGGCTGGAAA GCTTGCAGCC CCAGCCCGGG CCAGCCAGGT ACAGGAGGCC

GGACTGCAAC CGGTTGCTTC CCTCCCGTCG CGCCTGGCCG TCCCACGCTG CGCCGTCGCT

GCTGCCTCCT GGCGCCCCTG GGATTTTATA CGCACCTCTG AAACACGCTC CGCTCCGGCC

CCCGGTTCTT CTCCTTGCCT AGGGGTTGTT_TCCCAATAGA TACTGACTCC TTTAGAAGAT

CCAAAAACCA AACCAAA.ACA CCCCCTA�CC�GC�CCAAACA CCTGCTCTGG GGGCGCGGGG

GCTGCCAAAC AGAGACTAGA CGAAGGGAGT CAGATTTAGC GAAGCTCTTC GAGCTCCCAA

AGATTCGAAC ACTAACTCGC GCCCGTGGGC CGATGGAGGT TCTCCCTACT CCACTCCTTG

GTCCCCTTAA CTGGCTTCCG CCTCCTGGTC AATCACTGAG CAACCAGAAT GGTATCCTCG

ACCAGGGCCA CAGGCAGTGC TCGGCGGAGT GGCTCCAGGA GTTACCCGCT CCCTGCCGGG

CTTCGTATCC AAACCCTCCC CTTCACCCCT CCTCCCCAAA CTGGGCGCCA GGATGCTCCG

GCCGGAATAT ACGCAGGCTT TGGGCGTTTG CCCAAGGGTT TTCTTCCCTC CTAAACTAGC

CGCTGTTTTC CCGGCTTAAC CGTAGAAGAA TTAGATATTC CTCACTGGAA AGGGAAACTA

AGTGCTGCTG ACTCCAATTT TAGGTAGGCG GCAACCGCTT CCGCCTGGCG CAA.ACCTCAC

CAAGTAAACA ACTACTAGCC GATCGAAATA CGCCCGGCTT ATAACTGGTG CAACTCCCGG

CCACC�MC� G�GGACGTT CGCTTTCAGT CCCGACCTCT GGAACCC�A �MtGGCCACC

�CTTTCCCCA GTGACCCCAA GATCATGGCC ACTCCCCTAC CCGACAGTTC TAGAAGCAAG

AGCCAGACTC �GGGTGCAA AGCAAGGGTA TACGCTTCTT TGAAGCTTGA CTGAGTTCTT

TCTGCGCTTT CCTGAAGTT� ��CTCTT GGAGCCTACC TGCCCCTCCC TCCAAACCAC

TCTTTTAGAT TAACAACCc� ATCTCTACTC CCACCGCATT CGACCCTGCC CGGACTCACT

GCTTACCTGA ACGACTCTCC A�rGAGXCGA�GCTCCCACA CTGGCGAAGG CCAAGAAGGG

GAGGTGGGGG GAGGGTTGTG CCACACCGGC CAGCTGAGAG CGCGTGTTGG GTTGAAGAGG

AGGGTGTCTC CGAGAGGGAC GCTCCC�C�G AGc��1� A�CCCAGt1T!G CGAGG��

Cc�#{235}KA�GGAGC AGCGCGCGCT GCCTGGCCGG GCTTGGGCTG CTGAGTGAAT GGAGCGGCCG

AGCCTCCTGG CTCCTCCTCT TCCCCGCGCC GCCGGCCCCT CTTATTTGAG CTTTGGGAAG

CTGAGGGCAG CCAGG�AGCT GGGGTAAGGA GTTCAAGGCA GCGCCCACAC CCGGGGGCTC

TCCGCAACCC GACCGCCTGT CCqCTCCCCC_AqT�C�CGCi� CTCCCTCCCA_CCTACTCATT

CACCCACCCA CCCACCCAGA GCCGGGACGG CAGCCCAGGC �

TCGCCGCGAT CCTGGACTTC TCTTGCTGCA GGACCCGGCT TCCACGTGTG TCCCGGAGCC

GGCGTCTCAG CACACGCTCC GCTCCGGGCC TGGGTGCCTA CAGCAGCCAG AGCAGCAGGG

AGTCCGGGAC CCGGGCGGCA TCTGGGCCAA GTTAGGCGCC GCCGAGGCCA GCGCTGAACG

TCTCCAGGGC CGGAGGAGCC GCGGGGCGTC CGGCTCTGAG CCTCAGCAAA TGGGCTCCGA

CGTGCGGGAC CTGAACGCGC TGCTGCCCGC CGTCCCCTCC CT...

I

I-

�-200


7 A. Memisoglu and K. Call, unpublished results.

A-1232

-1200-1140-1080

-1020

- 960- 900

- 840

- 780- 720

- 660- 600- 540- 480

- 420

- 360- 300

- 240- 180- 120- 60+ 1

+ 61+ 121+ 181+ 241+ 301

+ 361�1� 421

+ 481+ 541+ 601+ 661+ 721

+ 781

+ 841+ 901+ 961

+s1

��

‘- w LO��c1)O�

ff8� - 700. -600

#{149}�-500

p -400

Fig. 6. DNA sequence analysis ofthe human V/fl 5’ flanking DNA sequence and Si nuclease analysis. A, WT1 genomic DNA sequence from + 1 232 to -1002bp. The major ORF encoding WT1 protein is underlined, starting at position +950 bp. Transcription initiation sites observed at positions +1, +71, +200, and+556 bp (±5 to 10 bp) are marked with bent arrows. No distinction has been made between degree of use oftranscription initiation sites since use frequency

differs between cell lines. DNA sequences similar to Spl/GC boxes (GGCGGG) are shown in stippled boxes. A cluster of 5 sequences with similarities to theEGR-1 binding site (GCGGGGGCG) between +388 to +425 bp are shown in open boxes. Two other potential EGR-1 binding sites are located at +624 to +632

and +705 to +713 bp (open boxes). A sequence corresponding to a TRE element (AP-1 binding site) is highlighted (diagonal slashed box). Sequences with

similarities to the serum response element core consensus sequence in the vicinity of the + 1 transcription initiation site are shown in dark stippled boxes. Thefollowing minor differences were noted in our sequence data compared to Hofmann et al. (48): + an extra G (-608 bp), GC (-558, -557) versus an N, 1 lessC at + 253. Data reported by Gessler and Bruns 59), which extends as far 5’ as + 1 37 bp, agrees with our data. The GENBANK accession number for our sequence

data is U06486. B, Si nuclease analysis. WT1 mRNA transcripts in K562 and CEM cells were analyzed by Si nuclease mapping as described in “Materials and

Methods.” Following Si treatment, the products were electrophoresed on a 6% polyacrylamide-8M urea gel and autoradiographed. The protected fragments (l-lV)observed are marked on the left side of the figure. The radiolabeled 1 00-hp ladder size standards are shown in the right lane with molecular weights indicated.

growth, since we have found that a variety of conditionsand agents which inhibit proliferation have no effect on thelevel of WTJ mRNA. This is supported further by our North-em blot analyses which showed that the total steady-statelevel of WTJ transcripts in cell cycle-fractionated K562cells remains relatively constant.7 Conversely, these obsen-vations indicate that if WT1 is regulated during the cellcycle, it is not modulated at the level of total WTJ tran-scripts. The very rapid down-regulation of WT1 mRNA inresponse to TPA also argues against this decrease resultingfrom growth inhibition. Our experiments with bryostatmn-1show that WT1 mRNA is down-regulated in response toTPA only when megakaryocytic differentiation is induced

subsequently. It will be of future interest to determinewhether WTJ also is decreased during granulocytic andmonocytic differentiation of K562 cells.

The results reported herein are consistent with findings

that the level of WT1 mRNA is correlated inversely withdifferentiation status during kidney development (1 0) and inWilms’ tumors (30-32) and leukemias (14, 15). However,

our findings are the first report, to our knowledge, to: (a)demonstrate a decrease in WT1 expression during the in-

duction of differentiation in a cell culture model, and (b)investigate the kinetics and mechanisms of this down-meg-ulation. These findings further support a mole of the WTJgene product in hematopoietic cells and suggest that the

K562 cell line will be a valuable system for investigating thefunction of the WT1 gene during differentiation.

The expression of INTl and its down-regulation during

differentiation of K562 cells is predicted to be functionallysignificant based collectively on several lines of evidence:(a) as previously discussed, the literature strongly supportsmoles for WTJ in both proliferation and differentiation pro-cesses; (b) the findings that a large number of cells ofhematopoietic origin express WTJ and this expression is


8 C� Lindberg and K. Call, unpublished results.

inversely correlated with differentiation status (2, 14, 15)suggest a role for W11 in hematopoietic differentiation; (c)there is evidence that K562 cells express fully, or at leastpartially, functional protein. In the K562 cell line, normal-sized w-ri products are observed in Northern blots, reversetranscription polymemase chain reactions, RNase protectionassays, and Western blots (2, 23).8 Furthermore, no muta-(ions have been observed in the WT1 coding sequencesexamined in K562 cells (23).8

The data also indicated that the decrease in WTJ mRNAduring induction of differentiation by TPA occurs, at leastpartially, by a posttnanscniptional mechanism. To date,there have been no data reported on a regulatory mecha-nism for decreased WTJ expression during differentiation ofany other cells. An intriguing possibility is that this post-transcriptional down-regulation occurs by induction of en-dogenous antisense WT1 mRNA. During differentiation ofMEL cells, p53 mRNA is down-regulated, and this eventoccurs posttranscniptionally (45). Interestingly, there is evi-dence that the down-regulation of p53 in these cells ismediated by induction of endogenous antisense p53 in-tronic mRNA (46). There has been a preliminary report of anantisense WT1 mRNA (47), which lends credence to oursuggestion that WT1 mRNA is regulated by an antisensemechanism during differentiation. This will be important totest, since expression of endogenous antisense mRNA hasnot been documented widely as a mechanism of eukaryoticgene regulation.

To gain insights into potential mechanisms of WTJ tran-scriptional regulation, we analyzed the 5’ flanking region ofthe WT1 gene for regulatory elements and transcriptioninitiation sites. Our findings of multiple transcription initi-ation sites and qualitative differences in the use of thesesites between cell lines, as well as the alternative splice sites(6), underscore the complexity of the WT1 transcripts inhuman cells. Multiple transcription initiation sites havebeen associated with long 5’ untranslated regions, as ex-hibited by WT1 mRNA. A comparison of the human tran-scniption initiation sites shows partial correspondence withpublished human (48) and mouse (10) fetal kidney data.The +200 bp WT1 initiation site in the K562 and CEM celllines corresponds to the mouse +1 transcription initiationsite. The human WT1 +556 bp transcription initiation sitein CEM cells corresponds to the major transcription start sitein mouse fetal kidney (10) and a cluster of sites in humanfetal kidney (48). The two furthest 5’ human WT1 transcnip-(ion initiation sites (+1 and +71 bp; Fig. 6A) were notreported in human (48) and mouse (1 0) kidney. These up-stream transcription initiation sites may exist in human andmouse kidney but would not have been detected sinceneither the human nor mouse probes assayed extended thisfar upstream. The two most 5’ initiation sites also could bespecific to hematopoietic cells.

In human WT1 transcribed sequences, there are twoshort ORFs upstream of the start of the major WT1 ORF atposition +950 bp (Fig. 6A). The first short ORF in thehuman begins at position +25 bp and extends for 90 nude-otides to +1 1 4 bp. The second short ORF extends for 99nucleotides from +469 to +567 bp. Neither of these read-ing frames have marked similarities to known peptide se-quences or domains. However, a comparison of the human

and mouse DNA sequence showed stretches of markedconservation starting at +137 bp in the human and con-tinuing through the WTJ coding sequence. (Comparisonupstream of human -40 bp was precluded given the lack ofsequence data in the mouse.) There is no mouse ORF in theregion corresponding to the first short human WT1 ORF(+25 to +114 bp). Conversely, there is not a human WTJ

ORF corresponding to the first short ORF observed in themouse, despite similarities at the nucleotide level and acomparable EGR1 binding site in this region in both spe-cies. The region of the second short human WT1 ORF,however, is highly conserved at the nucleotide level (83%)with mouse sequence. This ORF is 33 amino acids in thehuman and 19 amino acids in the mouse, of which the first10 amino acids are identical. These similarities suggestpotential functional significance.

Within our study, we also began to assess whether theremay be regulatory interactions between WTJ and othergenes implicated in growth and differentiation of K562cells. For example, collectively the observations that IGF-llis implicated in fetal kidney development (reviewed in Ref.49), is overexpressed in Wilms’ tumors (50), and possessesEGR-1 binding sites in its promoter, led to the finding thatw-ri m� capable of regulating the transcription of IGF-lIpromoter sequences (1 8). We reasoned that analysis of theregulation of WT1 and other potentially key genes duringK562 cell differentiation would lead to additional testablehypotheses regarding the regulatory interactions and role ofthe WTJ product.

There are indications that WTJ protein can act as atranscriptional repressor of the TGFf31 and PDGFB genes.There is an EGR1 (WT1) binding site and other similarsequences in the TGFf31 (42) and PDGFB promoters (43).Transcriptional regulation ofTGFf3l by WT1 protein also issupported by in vivo expression patterns. During kidneydevelopment, TGF�1 is expressed in mesenchymal regions(51) in a temporal and spatial pattern similar to the WT1protein (8, 10, 52). Both TGFf31 and WT1 have been hy-pothesized to play a role in embryonic development and inmediating mesenchyme-epithelial interactions (51 , 52).

The time course data (Fig. 4, A and B) and the WT1mRNA half-life data (Fig. 5) are consistent with WT1 actingas a transcriptional repressor of the TGFf31 and PDGFBgenes. However, if WT1 is a repressor of TGF�1 , it is onlypartially effective, since there are significant basal levels ofTGFf31 mRNA in K562 cells. It also is possible that WT1 isnot a transcriptional repressor of TGF�3i in K562 cells andthat the increased level of TGF�1 mRNA during TPA treat-ment is mediated solely by an activation mechanism via theTRE element in the TGF�31 promoter (44). Further expeni-ments are being undertaken to discern these possibilities.

The product of another tumor suppressor gene, p53, alsohas been implicated in the differentiation of K562 cells (53).The p53 allelesin the K562 cell line have been reported tobe functionally mutant (54). Stable transfection and expres-sion of p53 in K562 cells results in increases in parametersof erythroid differentiation (53). Of relevance, it has beenreported that there are physical interactions between p53and WT1 protein which have functional consequences (22).Given these observations, it will be of interest to investigatepotential interactions between WT1 and p53 protein duringdifferentiation and the functional significance of any suchevents in the appropriate systems.

w-ri protein may have multiple roles in growth anddifferentiation which may be complex, temporal, and de-


pendent on cell or tissue type. Although sufficient data havenot been reported to propose comprehensive models, ex-isting evidence suggests that WT1 protein may regulateproliferation of (relatively) undifferentiated cells by repress-ing expression of IGF-ll, IGFIR, or other genes involved instimulating growth or genes necessary for induction of dif-ferentiation. Given that WTJ protein can act as a transcnip-tional activator (21 , 22), it also could be a positive regulatorof genes inhibiting cell growth or differentiation. Rb hasbeen proposed to retain cells in an undifferentiated state(“status quo”) until it decreases to a threshold which facil-itates differentiation (55). This model also fits the data forw-ri. Further functional investigations, such as antisenseand sense expression experiments, should aid in under-standing the roles of WT1 protein in differentiation andproliferation in K562 and other cell lines which express thisgene. In addition, differentiation investigations may shedlight on how mutations in this gene contribute to cancer anddevelopmental abnormalities. The data reported herein pro-vide insights into the kinetics and mechanisms of WT1regulation during K562 cell differentiation and suggest thatthis line will be a valuable system for analyzing the roles ofWTJ in differentiation and proliferation.

Materials and Methods

Cell Culture. The human K562 (chronic myelogenous leu-kemia) and CEM (acute lymphoblastic leukemia) cell lines(ATTC Repository) were grown in RPMI 1 640 supplementedwith 10% (v/v) heat-inactivated HS containing penicillin(100 units/mI) and streptomycin (100 pg/mI) and incubatedat 37#{176}Cin a 5% CO2 atmosphere. In all experiments, cellswere used in logarithmic growth phase and seeded at adensity of 1 x i0� cells/mi. Medium supplemented with0.5% HS was used in the serum-starvation experiments.

Differentiation Indudion and Measurement. The fol-lowing inducers and inhibitors of differentiation wereadded to the culture medium at the stated final concentra-(ions: 3 nM TPA (Sigma Chemical Co.) in DMSO, 1 mt�ibutynic acid-sodium salt (Sigma) in water, and 1 nt�.i or 100nM bryostatin-1 (LC Laboratories) in DMSO. Megakaryo-cytic differentiation was assayed in cells 6-48 h after TPAor mock treatment by fluorescent activated cell sorting(FACS) analysis on cell samples stained with a fluoresceinisoth iocyanate-conjugated mouse monoclonal gpIIla anti-body (Dako Corp.). Briefly, 3 x 10� cells were harvestedfrom each culture, washed three times in PBS, incubatedwith 0.5 ml ofa 1 :40 dilution ofthe antibody (41) for 30 mmat 4#{176}C,washed twice in PBS, and resuspended in 0.5 ml ofPBS for analysis on a cytofluorograph flow system #21 51using Cicero software (Cytomation). Data was expressed asthe mean fluorescence values and plotted as fluorescenceintensity (Log F) versus number of cells (n). Erythroid dif-fementiation was measured using a benzidine staining pro-cedure optimized for K562 cells (40). Benzidine base wasobtained from Sigma.

RNA Preparation and Northern Blots. Total RNA wasextracted from cellsusing the single-step acid guanidmnium-phenol-chloroform method (56). Fifteen pg of each RNAsample in ethidium bromide (0.5 pg/mI) were electropho-resed on a 1% agarose gel containing 0.22 M formaldehyde,20 m� 4-morpholinepropanesulfonic acid, 5 m�i sodiumacetate, and 1 mM EDTA. RNAs were transferred from thegel onto a nylon membrane (Gene Screen; NEN-Dupont)and UV-cross-linked to the membrane. Membranes were

prehybnidized in 50% formamide, 1 % sodium dodecyl sul-fate, 1 M sodium chloride, and 10% dextran sulfate at 42#{176}C.DNA fragments were madiolabeled with [32P]dCTP by the

random priming method using reagents from Pharmaciaaccording to the supplier’s instruction. Membranes werehybridized with prehybmidization solution plus 4 x 1 �6

cpm of denatured 32P-labeled probe. After 18-48 h ofhybridization, membranes were washed twice for 5 mm atroom temperature in 2 X standard saline citrate, 1 0-30 mmat 60#{176}Cin 2X standard saline citrate/1% sodium dodecylsulfate, and exposed to X-ray film with an intensifyingscreen at -80#{176}Cfor 1-7 days. Densitometnic analysis ofautoradiograms was performed on a Biolmage Perfect Scan3cx densitometer (Millipore) using the Band Analysisprogram.

cDNA Probes. A 1 .8-kb EcoRl fragment correspondingto the w-ri gene was isolated from the human WT33 cDNAclone (2). A 2.1-kb EcoRI fragment was isolated from thephTGFf3-2 plasmid clone corresponding to a human TGF�31

cDNA inserted into pBR327 (ATCC). A 2.1 -kb BamHl frag-ment of the human PDGFB (c-sis) cDNA was isolated fromthe pcDVl clone (ATCC). The egr-1 probe was a 3.2-kbcDNA fragment derived from the mouse zif268 clone(ATCC). The f3-actin probe used was a 0.5-kb PstI mouse�-actin cDNA insert cloned in the pGEM-3 vector. Thehuman 36B4 nibosomal protein cDNA probe was isolatedas a 0.7-kb Pstl fragment cloned in pBR322.

DNA Sequencing. DNA sequencing was performed bythe dideoxy chain termination method using double-strandtemplates with Sequenase reagents (USB Corp., Cleveland,OH). Genomic fragments corresponding to the 5’ flankingregions of w-ri were subcloned into pUC1 9 or pBluescniptvectors. Oligonucleotide primers for pBluescnipt were ob-tamed from New England Biolabs and primers correspond-ing to WT1 cDNA and genomic sequences were generatedby standard methods. DNA sequencing of both strands ofgenomic DNA was performed. Sequencing reactions wereelectrophoresed on 6% polyacrylamide gels, dried, andautoradiographed. The transcription factors database (57)was used to search for DNA regulatory elements.

Si Nuclease Analysis. 51 nuclease assays were per-formed with minor modifications of a method of Greeneand Struhl (58). End-labeled WTJ DNA probe was prepared

as follows. A genomic WT1 fragment corresponding to4.5-kb of WT1 5’ flanking and WT1 gene sequences wascloned into the BamHl site of pUC1 9. DNA from this sub-clone (p55B) was restriction digested with PpuMl, treatedwith calf alkaline phosphatase, radiolabeled with[‘y-32P]ATP, and restriction digested with Sail which cleaveswithin the polylmnker. The 1 .5-kb DNA fragment encom-passing the 5’ untranslated region and upstream WT1 DNAsequences (-763 to +766 bp) was isolated from 0.8% lowmelting temperature agarose and purified using the Gene-Clean method (BiolOi, La Jolla, CA). Total RNA (100 pg)isolated from K562 cells was ethanol precipitated with ma-diolabeled probe (1 00,000 cpms), resuspended in hybrid-ization buffer [80% formamide, 40 m�s PIPES (pH 6.4), 400mM NaCI, and 1 mM EDTA], denatured (90#{176}C,10 mm), andannealed overnight at 55#{176}C.Subsequently, samples weredigested with Si nuclease (300 units, 30#{176}C,30 mm), etha-nol precipitated, electrophoresed along with end-labeledsize markers on a 6% polyacrylamide-8 M urea gel, andautomadiographed.


AcknowledgmentsWe thank A. Memisoglu for helpful discussions and comments. We thankT. Nui for assisting with pilot experiments and A. Imrich for performingcytofluorographic analysis. We acknowledge Dr. S. May (Johns Hopkins

Oncology Center, Baltimore, MD) for a generous initial gift of bryostatin-1.

Note Added in ProofSekiya et al. (Blood, 83: 1 876-i 882, 1 994) have reported that V/Il mRNAis down-regulated similarly in HL-60 cells during granulocytic and mono-

cytic differentiation.

Referencesi . Matsunaga, E. Genetics of Wilms’ tumor. Hum. Genet., 57: 23 1-246,i98i.

2. Call, K. M., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber, D. A.,Rose, E. A., KraI, A., Yeger, H., Lewis, W. H., Jones, C., and Housman, D. E.Isolation and characterization of a zinc finger polypeptide gene at the humanchromosome 1 1 Wilms’ tumor locus. Cell, 60: 509-520, 1990.

3. Gessler, M., Poustka, A., Cavenee, W., Neve, R., Orkin, S., and Bruns,

G. A. P. A zinc finger gene identified by chromosome jumping. Nature(Lond.), 343: 774-778, 1990.

4. Morris, J. F., Madden, S. L., Tournay, 0. E., Cook, D. M., Sukhatme, V. P.,and Rauscher, F. J., Ill. Characterization ofthe zinc finger protein encoded bythe WT1 Wilms’ tumor locus. Oncogene, 6: 2339-2348, 1991.

5. Madden, S. L., Cook, D. M., Morris, J. F., Gashler, A., Sukhatme, V. P.,and Rauscher, F. J., Ill. Transcriptional repression mediated by the Wfl

Wilms’ tumor gene product. Science (Washington DC), 253: 1550-1553,1991.

6. Habem, D. A., Sohn, R. L., Buckler, A. J., Pelletier, J., Call, K. M., andHousman, D. E. Alternative splicing and genomic structure of the Wilms’

tumor gene wri. Proc. NatI. Acad. Sci. USA, 88: 961 8-9622, 1991.

7. Coppes, M. J., Campbell, C. E., and Williams, B. R. G. The role of WT1

in Wilms’ tumorigenesis. FASEB J., 7: 886-895, i993.

8. Pritchard-Jones, K., Fleming, S., Davidson, D., Bickmore, W., Porteous,D., Gosden, C., Bard, J., van Heyningen, V., and Hastie, N. The candidateWilms’ tumour gene is involved in genitourinary development. Nature

(Lond.), 346: 194-197, 1990.

9. Bove, K. E., and McAdams, A. J. The nephroblastomatosis complex andits relationship to Wilms’ tumor-a clinicopathological treatise. Perspect.Pediatr. Pathol., 3: 185-223, 1976.

10. Pelletier, J., Schalling, M., Buckler, A. J., Rogers, A., Haber, D. A., andHousman, D. Expression of the Wilms’ tumor gene Wfl in the murine

urogenital system. Genes Dev., 5: 1 345-1 356, 1991.

1 1 . Pelletier, J., Bruening, W., Li, F. P., Haber, H. A., Glaser, T., andHousman, D. E. WT1 mutations contribute to abnormal genital systemdevelopment and hereditary Wilms’ tumor. Nature (Lond.), 353: 431-434,i 991.

12. Pelletier, J., Bruening, W., Kashtan, C. E., Mauer, S. M., Manivel, J. C.,Striegel, J. E., Houghton, D., Junien, C., Habib, R., Fouser, L., Fine, R. N.,Silverman, B. 1., Haber, D. A., and Housman, D. Germ line mutations in theWilms’ tumor suppressor gene are associated with abnormal urogenitaldevelopment in Denys-Drash syndrome. Cell, 67: 437-447, 1991.

13. Kreidberg, J. A., Sariola, H., bring, J. M., Maeda, M., Pelletier, J.,Housman, D., and Jaenisch, R. Wil is required for early kidney develop-

ment. Cell, 74: 679-702, 1993.

14. Miwa, H., Beran, M., and Saunders, G. F. Expression of the Wilms’tumor gene (W�1) in human leukemias. Leukemia (Baltimore), 6: 405-409,1992.

15. Miyagi, T., Ahuja, H., Kubota, T., Kubonishi, I., Koeffler, H. P., andMiyoshi, I. Expression of the candidate Wilms’ tumor gene, WT1, in human

leukemia cells. Leukemia (Baltimore), 7: 970-977, 1993.

16. Rauscher, F. J., Morris, J. F., Tournay, 0. E., Cook, D. M., and Curran,T. Binding of the Wilms’ tumor locus zinc finger protein to the EGR-iconsensus sequence. Science (Washington DC), 250: 1 259-i 262, i990.

17. Bickmore, W. A., Oghene, K., Little, M. H., Seawright, A., vanHeyningen, V., and Hastie, N. D. Modulation of DNA binding specificityby alternative splicing of the Wilms’ tumor Wil gene transcript. Science(Washington DC), 257:235-237, 1992.

18. Drummond, I. A., Madden, S. L., Rohwer-Nutter, P., Bell, G. I.,Sukhatme, V. P., Rauscher, F. J., Ill. Repression of the insulin-like growthfactor II gene by the Wilms’ tumor suppressor Wil. Science (WashingtonDC), 257:674-678, 1992.

1 9. Gashler, A. L., Bonthron, D. T., Madden, S. L., Rauscher, F. J., Ill, Collins,T., and Sukhatme, V. P. Human platelet-derived growth factor A chain istranscriptionally repressed by the Wilms’ tumor suppressor gene. Proc. NatI.

Acad. Sci. USA, 89: 10984-10988, 1993.

20. Werner, H., Re, G. G., Drummond, I. A., Sukhatme, V. P., Rauscher,F. J., Ill, Sens, D., Garvin, A. J., LeRoith, D., Roberts, C. T., Jr. Increasedexpression ofthe insulin-like growth factor I receptor gene, IGF1R, in Wilms’tumor is correlated with modulation of IGF1R promoter activity by the WI!Wilms’ tumor gene product. Proc. NatI. Acad. Sci. USA, 90: 5828-5832,1993.

21 . Wang, z. ‘s’., Qui, Q-Q., and Deuel, T. F. The Wilms’ tumor geneproduct WI! activates or suppresses transcription through separate func-tional domains. J. Biol. Chem., 268: 9172-9175, 1993.

22. Maheswaran, S., Park, S., Bernard, A., Morris, J. F., Rauscher, F. J., III,Hill, D. E., and Haber, D. A. Physical and functional interaction betweenWT1 and p53 proteins. Proc. NatI. Acad. Sci. USA, 90: 5i00-5i04, 1993.

23. Telerman, A., Dodemont, H., Degraef, C., Galand, P., Bauwens, S., VanOostveld, T., and Amson, R. B. Identification of the cellular protein encodedby the human Wilms’ tumor (Wfl) gene. Oncogene, 7: 2545-2548, i992.

24. Alitalo, R., Makela, T. P., Koskinen, P., Andersson, L. C., and Alitalo, K.Enhanced expression oftransforming growth factor � during megakaryoblas-

tic differentiation of K562 cells. Blood, 71: 899-906, i988.

25. Alitalo, R. Induced differentiation of K562 leukemia cells: a model forstudies of gene expression in early megakaryoblasts. Leukemia Res., 14:501-5i4, i990.

26. Lozzio, C. B. and Lozzio, B. B. Human chronic myelogenous leukemiacell linewith positive Philadelphia chromosome. Blood, 45:321-334, 1975.

27. Andersson, L. C., Nilsson, K., and Gahmberg, G. C. K562-a humanerythroleukemia cell line. Int. J. Cancer Res., 23: 143-147, 1979.

28. Vainchenker, W., Testa, U., Guichard, J., Titeux, M., and Breton-Gorius,J. Heterogeneity in the cellular commitment of a human leukemic cell line.

BIoodCeIIs, 7:357-375, i98i.

29. Leary, J. F., Ohlsson-Wilhelm, B. M., Guliano, R., LaBella, S., Farley, B.,and Rowley, P. T. Multipotent human hematopoietic cell line K562: lineage-

specific constitutive and inducible antigens. Leukemia Res., 11: 807-81 5,

1987.

30. Yeger, H., Cullinane, C., Flenniken, A., Chilton-MacNeill, S., Campbell,C., Huang, A., Bonetta, L., Coppes, M. J., Thorner, P., and Williams, B. R. G.Coordinate expression of Wilms’ tumor gene correlates with Wilms’ tumorphenotypes. Cell Growth & Differ., 3: 855-864, 1992.

3i . Gerald, W. L., Gramling, T. S., Sens, D. A., and Garvin, A. J. Expressionofthe 1 1 p1 3 Wilms’ tumor gene, WT1, correlates with histologic category ofWilms’ tumor. Am. J. Pathol., 140: 1031-1037, 1992.

32. Miwa, H., Tomlinson, G. E., Timmons, C. F., Huff, V., Cohn, S. L.,Strong, L. C., and Saunders, G. F. RNA expression ofthe WT1 gene in Wilms’tumors in relation to histology. J. NatI. Cancer Inst., 84: 181-187, 1992.

33. Andersson, L. C., Jokinen, M., and Gahmberg, G. C. Induction of ery-throid differentiation in the human leukaemia line K562. Nature (Lond.),278: 364-365, i979.

34. Rutherford, T. R., Glegg, J. B., and Weatherall, D. J. K562 humanleukaemic cells synthesize embryonic haemoglobin in response to hemin.Nature (Lond.), 280: i64-i65, i979.

35. Tetteroo, P. A., Massaro, F., Mulder, A., Schreuder-van Gelder, R., andvon dem Borne, A. E. G. Megakaryoblastic differentiation of proerythroblas-tic K562 cell line cells. Leukemia Res., 8: 197-206, i 984.

36. Alitalo, R., Partanen, J., Pertovaara, L., Holtta, E., Sistonen, L., Anderson,L., and Alitalo, K. Increased erythroid potentiating activity/tissue inhibitor ofmetalloproteinase and jun/fos transcription factor complex characterize tu-mor promoter-induced megakaryoblastic differentiation of K562 leukemiacells. Blood, 75: 1974-1987, 1990.

37. Colamonici, 0., Trepel, J. B., and Neckers, L. A. Megakaryocyte differ-entiation: studies at the molecular level using the K562 cell line. in: R.Levine, N. Williams, J. Levin, and B. Evatt (eds.), Megakaryocyte Develop-ment and Function, pp. i87-i9i. New York: Alan R. Liss, Inc., 1986.

38. Alitalo, R., Andersson, L. C., Betsholtz, C., Nilsson, K., Westermark, B.,Heldin, C-H., and Alitalo, K. Induction ofplatelet-derived growth factor geneexpression during megakaryoblastic and monocytic differentiation of humanleukemia cell lines. EMBO J., 6: 1213-1218, 1987.

39. Makela, T. P., Alitalo, R., Paulsson, Y., Westermark, B., Heldin, C-H.,and Altialo, K. Regulation of platelet-derived growth factor gene expressionby transforming growth factor �3 and phorbol ester in human leukemia celllines. Mol. Cell. Biol., 7: 3656-3662, 1987.

40. Wanda, P. E., Lee, L. T., and Howe, C. A spectrophotometric method formeasuring hemoglobin in erythroleukemia cells (K562). J. Histochem.Cytochem., 29: 1442-1 444, 1981.

686 Down-regulation of WTi mRNA during Differentiation

59. Gessler, M., and Bruns, G. A. P. Sequence of the WT1 upstream regionincluding the Wit-i gene. Genomics, 17: 499-501 , 1993.

41 . Hocevar, B. A., Morrow, D. W., Tykocinski, M. L., and Fields, A. P.Protein kinase C in human erythroleukemia cell proliferation and differen-tiation. J. Cell. Sci., 101: 67i-679, i992.

42. Kim, S-i., Glick, A., Spomn, M., and Roberts, A. B. Characterization of thepromoter region of the human transforming growth factor-ui gene. J. Biol.Chem., 264:402-408, i989.

43. Ratner, L., Thielan, B., and Collins, T. Sequences of the 5’ portion of thehuman c-sis gene: characterization of the transcription promoter and regu-lation of expression of the protein product by 5’ untranslated mRNA se-quences. Nucleic Acids Res., 15: 601 6-6036, 1987.

44. Kim, S-J., Denhez, F., Kim, K. Y., Holt, J. T., Sporn, M. B., and Roberts,

A. B. Promoter sequences of the human transforming growth factor-ni generesponsive to transforming growth factor-�3i autoinduction. J. Biol. Chem.,264: 19373-i9378, i989.

45. Khochbin, S., Principaud, E., Chabanas, A., and Lawrence, J-J. Earlyevents in murine erythroleukemia cells induced to differentiate. Accumula-tion and gene expression of the transformation-associated cellular proteinp53. J. Mol. Biol., 200: 55-64, 1988.

46. Khochbin, S., and Lawrence, J-J. An antisense RNA involved in p53mRNA maturation in murine erythroleukemia cells induced to differentiate.EMBO J., 8: 41 07-41 14, 1989.

47. Campbell, C., Gurney, A. L., Hewitt, J., Raychaudhuri, B., Kuriyan, N.,Rackley, R., Coppes, M. J., and Williams, B. R. G. Regulation of expressionof the WT1 locus. Proc. Am. Assoc. Cancer Res., 34: 541, 1993.

48. Hofmann, W., Royer, H-D., Drechsler, M., Schneider, S., andRoyer-Pokora, B. Characterization of the transcriptional regulatory region ofthe human WT1 gene. Oncogene, 8: 3123-3132, 1993.

49. Han, V. K. M., and Hill, D. J. The involvement of insulin-like growthfactors in embryonic and fetal development. In: P. N. Schofield (ed), TheInsulin-like Growth Factors, pp. 1 78-220. Oxford: Oxford University Press,1992.

50. Reeve, A. E., Eccles, M. R., Willins, R. J., Bell, G. I., and Millow, L. J.Expression of insulin-like growth factor-Il transcripts in Wilms’ tumour.Nature (Lond.), 317: 258-260, i 985.

5i . Lehnert, S. A., and Akhurst, R. J. Embryonic expression pattern of TGF �type-i RNA suggests both paracrine and autocrine mechanisms of action.

Development (Camb.), 104: 263-273, 1988.

52. van Heyningen, V. and Hastie, N. D. Wilms’ tumor: reconciling genetics

and biology. Trends Genet., 8: i 6-2i , 1992.

53. Feinstein, E., Gale, R. P., Reed, J., and Canaani, E. Expression of thenormal p53 gene induced differentiation in K562 cells. Oncogene, 7:1853-1857, 1992.

54. Law, J. C., Ritke, M. K., Yalowich, J. C., Leder, G. H., and Ferrell, R. E.The p53 gene is mutationally inactivated in the human erythroid leukemicK562 cell line. Proc. Am. Assoc. Cancer Res., 34: 537, 1993.

55. Yen, A., Chandler, S., Forbes, M. E., Fung, Y-K., T’ang, A., and Pearson,R. Coupled down-regulation of the RB retinoblastoma and c-myc genesantecedes cell differentiation: possible role of RB as a “status quo” gene.Eur. J. Cell Biol., 57: 210-221, i992.

56. Chomezynski, P., and Sacchi, N. Single-step method of RNA isolationby acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.Biochem., 162: 156-159, 1987.

57. Gosh, D. A relational database of transcription factors. Nucleic AcidsRes., 18: 1749-1756, i990.

58. Greene, J. M., and Struhl, K. Analysis of RNA structure and synthesis.in: F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology,pp. 4.6.i-4.6.i3. John Wiley and Sons, Inc., 1988.

wilms’tumorgene, wt1, mrna isdownregulated...

Documents