MOLECULAR AND CELLULAR BIOLOGY, Aug. 1988, p. 3526-3531 Vol. 8, No. 80270-7306/88/083526-06$02.00/0Copyright © 1988, American Society for Microbiology

The NF-KB-Binding Site Mediates Phorbol Ester-InducibleTranscription in Nonlymphoid CellsBARBARA NELSEN, LARS HELLMAN,t AND RANJAN SEN*

Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University,Waltham, Massachusetts 02254-9110

Received 21 January 1988/Accepted 14 April 1988

The mouse immunoglobulin K light-chain enhancer can interact with at least three independent nuclearproteins. One of these proteins, NF-KB, is constitutively present only in nuclear extracts derived from B cellsand plasma cells. A DNA-binding protein with the same sequence specificity (and therefore presumed to beNF-KB itself) can be induced in pre-B cells, T cells, and nonlymphoid cells by phorbol 12-acetate-13-myristate(PMA); however, it is not clear whether the induced factor can activate transcription in nonlymphoid cells asNF-KB does in B cells. In this paper we show that multimerization of a fragment of the mouse K enhancer thatcarried only the binding site for NF-KB behaved like a B-cell-specific regulatory element. Furthermore, thisunit served to activate transcription in nonlymphoid cells after treatment with PMA (but not with cyclic AMPderivatives), and the kinetics of transcription activation correlated well with the kinetics of factor induction.Thus, the induced DNA-binding activity appeared to be functionally indistinguishable from that of NF-KB.

B-cell-specific transcription of the immunoglobulin genesis governed in part by tissue-specific enhancers located inthe heavy-chain (4, 11, 12, 22, 24) and the kappa (K)light-chain loci (26, 27). Most of the activity of the heavy-chain enhancer can be located in a 220-base-pair (bp) Hinflfragment that lies within the originally identified 700-bpXbaI-EcoRI fragment. The smaller fragment has the abilityto interact with at least four independent nuclear factors (3,25, 30, 35), of which three are found in extracts derived froma wide variety of cell types. One factor (NF-A2) that binds toa conserved octanucleotide sequence (ATTTGCAT) appearsto be restricted in expression to B and T cells only (18, 33)and is therefore a good candidate to confer the observedtissue specificity of the enhancer. The K light-chain enhanceris organized in a similar fashion and can interact withtissue-specific and nonspecific factors. Deletion analysis ofthe K enhancer has shown that 30% of the activity is locatedin a 200-bp DdeI fragment within the JK-CK intron (28).Analysis of nuclear protein-binding sites by the electropho-retic mobility shift assay has shown that this segment bindsat least three proteins in a sequence-specific manner (30).One of these proteins, NF-KB, binds to the B site located inthe 5' DdeI-HaeIII fragment of the K enhancer and isconstitutively present only in extracts derived from cells thatrepresent the B-cell and plasma cell stages of B-lymphocytedifferentiation (30). This distribution correlates exactly withthose cells in which K genes are normally transcribed,suggesting that this protein may be a critical determinant ofK enhancer function. Recently, point-mutational analysis hasshown that elimination of the B site decreases K enhancerfunction to background levels in transient transfections intoB cells (21). Although NF-KB is constitutively present onlyin B cells, it or a protein with the same DNA-bindingspecificity can be induced in a variety of cell types, includingfibroblasts, by the action of active phorbol esters (31).However, it was not clear whether the protein induced innonlymphoid cells could also activate transcription analo-

* Corresponding author.t Present address: Department of Immunology, The Biomedical

Center, Box 582, S-751 23 Uppsala, Sweden.

gous to that by NF-KB. We show here that this is indeed thecase, making it very likely that the induced protein is thesame as that found constitutively in B lymphocytes.

MATERIALS AND METHODS

Cell lines and culture. HeLa is a human cervical carcinomacell line. Stably transfected HeLa cell lines KCAT-1,KCAT-2, KCAT-4, and SPCAT were grown in Dulbeccomodified Eagle (DME) medium supplemented with 10% calfserum. HAFTL is a pre-B-cell line grown in RPMI mediumcontaining 5% inactivated fetal calf serum and 0.0004%P-mercaptoethanol. This cell line does not synthesize immu-noglobulin heavy chains and undergoes DH to JH rearrange-ment in culture (1). Both the simian virus 40 (SV40) enhancer(data not shown) and the ,u heavy-chain enhancer are activein this cell line. AJ9 is a Lyl+ B-cell line derived from A/Jmice (6) which was grown in RPMI medium supplementedwith 5% inactivated fetal calf serum, 5% calf serum, andP-mercaptoethanol. S194 is a murine myeloma cell linegrown in DME medium supplemented with 5% inactivatedfetal calf serum and 5% (donor) calf serum.

Plasmids. pSPCAT was used both as a negative control fortransfection experiments and as the parent plasmid forgenerating the rest of the plasmids. It contains the bacterialchloramphenicol acetyltransferase (cat) gene and the SV40early promoter, but lacks the SV40 enhancer. It was gener-ated by excising the BglII-XbaI fragment of pAlOCAT andcloning it into pSP64 cut with BamHI and XbaI. The testplasmid p(K3)3CAT, containing a trimer of the NF-KB-binding site (K3 fragment [30]) was made by excising theDdeI-HaeIII fragment of the K enhancer, treating it withKlenow fragment of DNA polymerase to create blunt ends,and then ligating it into pSPCAT cut with SmaI. p,uECATwas used as a positive control for cat expression in B-lymphoid cell lines. It contains the 220-bp Hinfl-Hinfl frag-ment retaining full IL enhancer activity cloned into the SmaIsite of pSPCAT. The positive control for nonlymphoid celltypes, pSV2CAT, contains the SV40 enhancer as well as thepromoter.

Transfections and cat expression analysis. Transient trans-fections were done into tissue culture cells representing

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NF-KB-BINDING SITE IN NONLYMPHOID CELLS 3527

different stages of B-cell development and nonlymphoid cells(HeLa) with the plasmids p(K3)3CAT, pSPCAT, and eitherp,uECAT or pSV2CAT. A total of 2 x 107 lymphoid cellswere transfected with 10 ,ug of supercoiled plasmid by theDEAE-dextran procedure. At 44 to 48 h after transfection,the cells were washed three times with phosphate-bufferedsaline and suspended in 100 ,ul of 0.25 M Tris, pH 7.8. Cellextracts were generated by carrying out three freeze-thawcycles followed by centrifugation at 4°C to spin out thedebris. Transfections of 100-mm dishes of subconfluentHeLa cells (106 cells per dish) with 2 ,ug of DNA was alsoperformed by the DEAE-dextran method. After 44 to 48 h,the plates were washed twice with phosphate-buffered sa-line, allowed to sit for 5 min at room temperature in 1 ml ofTris-EDTA-NaCl (0.04 M Tris hydrochloride [pH 7.4], 1 mMEDTA, 0.15 M NaCI). The cells were scraped off with arubber policeman and pelleted at 4°C. The cells were sus-pended and extracts were generated as described above forlymphoid cells. Total protein content of extracts was deter-mined by using the Bradford assay (Pierce), and equalamounts of protein were analyzed for CAT activity. CATenzyme assays were typically performed with 300 ,ug ofprotein and 0.1 p.Ci of [14C]chloramphenicol for 2 h at 37°C.After extraction with ethyl acetate, the acetylated productswere resolved by thin-layer chromatography (TLC) withSilica gel TLC plates with a 95% chloroform-5% methanolmobile phase. Following autoradiography, the results werequantified by excising the acetylated and unacetylated formsof chloramphenicol and determining the amount of radioac-tivity by liquid scintillation counting.

Stable HeLa cell lines carrying either the p(K3)3CAT orthe pSPCAT plasmids were generated by linearizing theplasmids at the PstI site in the polylinker and cotransfectingwith linearized pSV2Neo (at a ratio of 10:1) by electropora-tion. Cells were allowed to recover for 24 h and then placedin selective medium containing G418 (1 mg/ml). Resistantclones were picked and propagated in G418 for 3 to 4 weeks.Southern blot analysis showed that the cat gene was intact inthese transfectants and present in about 7 to 8 copies per cell(data not shown). Phorbol ester induction of the stablyintegrated plasmid was carried out at a concentration of 100ng/ml after the cells were removed from selective medium. A100-mm dish of confluent cells was split 1:5 and treated withphorbol 12-acetate-13-myristate (PMA) approximately 20 hlater (at 2 x 106 cells per dish). Twenty hours after PMAtreatment, extracts were prepared, and the CAT activity inthese extracts was analyzed as described above.RNA analysis. Primer extension analysis was performed

on RNA isolated by using the guanidinium thiocyanateprocedure from cells that had been treated with PMA. TheCAT primer used was a 25-bp oligonucleotide derived fromthe 5' untranslated region of the CAT transcript. The con-trol, endogenous actin mRNA, was analyzed by using a26-bp oligonucleotide derived from bases +109 to +134 ofthe human cytoplasmic ,-actin gene. The extended productswere quantified by densitometric scanning of the autoradio-gram.

RESULTS

c3 fragment enhances transcription in a tissue- and stage-specific manner. To focus on the role of NF-KB as a tran-scriptional activator, we asked whether a DNA fragmentcontaining the B site alone could serve to activate transcrip-tion in a tissue-specific manner. A trimer of the DdeI-HaeIIIfragment (K3 fragment [30]) of the K enhancer was cloned

upstream of the bacterial cat gene being transcribed froman enhancerless SV40 early promoter. This plasmid [p(K3)3CAT, Fig. 1A) was transfected into tissue culture cellsrepresenting different stages of B-cell development andnonlymphoid cells. In each experiment, we also transfectedplasmids that would serve as a negative control (pSPCAT,carrying only the SV40 early promoter sequences) or apositive control (p,uECAT, which has the full p. enhancercloned into pSPCAT or pSV2CAT). Levels of CAT enzymeactivity were assayed in extracts derived from the cells 48 hafter transfection and compared among the three plasmids.The K3 trimer enhanced transcription to an extent compa-

rable to that of the full p. enhancer itself in B cells (Fig. 1B,compare lanes 5 and 6 with lane 4) and plasma cells (Fig. 1B,compare lanes 8 and 9 with lane 7) but yielded only back-ground levels of activity in pre-B cells (Fig. 1B, comparelanes 3 and 1; lane 2 is the positive control with p,uECAT)and nonlymphoid cells (Fig. 1B, compare lanes 10 and 12;lane 11 is the positive control, pSV2CAT). This clearlyshowed that the K3 trimer was able to enhance transcriptionof the SV40 early promoter in a tissue-specific and stage-specific manner. Restriction of the activity of this syntheticregulatory element to only B cells and plasma cells, togetherwith the fact that NF-KB was the only factor known toassociate with this DNA fragment (see below), stronglysuggests that the effect was mediated by this factor.

Interestingly, in plasma cells the extent of enhancementobtained with the K3 trimer was much greater than thatobtained with the wild-type K enhancer, which, when as-sayed with the same promoter and gene combination,yielded only a fourfold enhancement (9; B. Nelsen, unpub-lished results). Thus, trimerization of a fragment of anenhancer can apparently lead to significantly higher activitythan with the enhancer itself. This observation is in contrastto those obtained by Gerster et al. (10), who tandemly linked6 copies of a 50-bp fragment of the p. enhancer and obtainedactivity equivalent to that of the full enhancer. However,multimerization of the complete p. enhancer has been shownto yield increased activity proportional to the copy number(17).

Phorbol ester-induced K3-binding protein activates tran-scription in nonlymphoid cells. To further characterize theinduction of NF-KB in nonlymphoid cells at a functional(transcriptional) level, we generated stable HeLa cell linescarrying either the p(K3)3CAT or the pSPCAT plasmid.Three transfectants carrying the p(K3)3CAT construct

(referred to as KCAT1, -2, and -4) and one line carryingpSPCAT (SPCAT) were picked for further analysis. Tocheck whether the transfected genes in KCAT lines could beinduced, the cells were treated with PMA for 24 h and CATenzyme activity was assayed in extracts made from the cells.All three KCAT lines were inducible approximately 7- to10-fold for CAT activity (Fig. 2, lanes 1 to 6). In contrast, theSPCAT line was not inducible at all (Fig. 2, lanes 7 and 8).Thus, under stimulatory conditions, the K3-binding proteinsinduced in these nonlymphoid cell lines could activatetranscription of the transfected gene. It is noteworthy thatthe inductions reported here were observed without priorserum starvation of the cells, a procedure which is oftenrequired to observe phorbol ester stimulation in HeLa cells(see, for example, references 2, 15, 16, 19, and 20). Thisprobably reflects the fact that the K3 DNA-binding activity isnormally undetectable in HeLa cells grown in 10% calfserum and is induced by phorbol ester treatment. In contrastto this, some other factors mediating transcription of PMA-inducible genes are found constitutively in HeLa cells (2, 5,

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3528 NELSEN ET AL.

AEco RI Bgl i1/Bam Hi Eco RI Xba I

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FIG. 1. Structures of plasmids and activity in cell lines representing different stages of B-lymphocyte development. (A) Structure of thep(K3)3CAT plasmid. pSPCAT is the parent plasmid that contains the SV40 early promoter without the enhancer. The K3 fragment is derivedfrom the mouse K enhancer and binds the B-cell-specific factor NF-KB. (B) K3 fragment confers B-cell-stage-specific activity in transienttransfection assays. p(K3)3CAT (lanes 3, 6, 9, and 12) and two control plasmids were transfected into cells representing different stages ofB-cell differentiation and human HeLa cells, followed by analysis of CAT enzyme activity 48 h later. The negative control for all experimentswas pSPCAT (lanes 1, 4, 7, and 10). The positive control for the B-cell transfections was p1LECAT, which contains the enhancer clonedinto pSPCAT (lanes 2, 5, and 8) or, for HeLa cells, pSV2CAT (lane 11). Cell lines are indicated at the bottom. The numbers above the lanesshow the fold stimulation of expression observed relative to that with pSPCAT, which was arbitrarily assigned a value of 1. All values are

the results of at least three independent experiments carried out in duplicate. The calculated standard deviation between experiments was lessthan 10%.

1 2 3 4 5 67 3

KCAT-1 KCAT--2 KCAT--- 4 SPCAT7.3 7.6 91

FIG. 2. Phorbol ester-induced CAT enzyme activity in stableHeLa cell transfectants. Three cell lines carrying stably integratedp(K3)3CAT plasmid (KCAT-1, -2, and -4) and one line carryingpSPCAT were analyzed for CAT enzyme levels in extracts preparedfrom unstimulated cells or those that had been stimulated with PMA(100 ng/ml) for 20 h. Assays were carried out in extracts derivedfrom untreated (-) and treated (+) cells. The numbers in bracketsbelow the cell lines refer to the extent of stimulation observedrelative to CAT levels present prior to PMA treatment in the sameline. Enzyme activities from stimulated and unstimulated cells wereassessed simultaneously in duplicate samples, and the numbersreported are the average of at least three independent experiments.CAT enzyme levels were normalized to total protein content.

19, 20). When the stable HeLa transfectants were stimulatedafter serum starvation, no further increase in the level ofinduction was observed.To test whether the transfected gene could be induced via

the protein kinase A pathway, we stimulated KCAT2 cellswith two different cyclic AMP (cAMP) analogs [dibutyrylcAMP and 8-(4-chlorophenylthio)-cAMP] in the presence of9-isobutyl-methylxanthine (a phosphodiesterase inhibitor) orwith forskolin (an adenylate cyclase activator) and 9-iso-butyl-methylxanthine. The experiments were repeated onserum-starved KCAT2 cells. However, under no conditionswas a significant increase in CAT enzyme levels observed(C. Jamieson and R. Sen, unpublished observations). Thus,at least in HeLa cells, NF-KB is apparently not activatablevia this pathway.

Kinetics of induction of CAT enzyme and RNA correlatewith factor induction in nonlymphoid cells. To determine thetime course for activation, KCAT2 cells were treated withPMA for different lengths of time and extracts were analyzedfor CAT activity. A detectably higher level of CAT enzymewas seen 3 h after stimulation (Fig. 3A, lane 2). The levelcontinued to increase, reaching a maximum at about 24 h(Fig. 3A, lane 5), and was detectably less after 31 h ofinduction (Fig. 3A, lane 6). It has been shown earlier thatPMA induction of NF-KB in 70Z cells reaches a maximumwithin 0.5 to 1 h and is barely detectable after 8 h ofstimulation (31). The continued presence ofPMA was there-fore proposed to downregulate NF-KB, perhaps by desensi-tization of the endogenous protein kinase C. The time courseof CAT enzyme induction is consistent with this behavior ofNF-KB. There was a latency period before any effect was

pSP64

MOL. CELL. BIOL.

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NF-KB-BINDING SITE IN NONLYMPHOID CELLS 3529

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FIG. 3. Time course of phorbol ester-induced CAT activity andmRNA in HeLa cell transfectant KCAT-2. (A) Time course of CATenzyme activity. KCAT-2 cells were induced with 100 ng of PMAper ml (in duplicate) for various times as indicated, and cell extractswere assayed for CAT activity as described. The fold inductionrelative to untreated cells is indicated in parentheses below eachline. In two independent sets of experiments, the standard deviationwas less than 10%. Care was taken to ensure that cells for thevarious time points were harvested at the same density. (B) Timecourse of cat mRNA induction. Primer extension analysis wasperformed on RNA isolated by using the guanidinium thiocyanateprocedure from cells that had been treated with PMA for varioustimes. The same transcription initiation site was observed in in vivoas in in vitro transcription reactions. Inset: lane 1, no induction;lanes 2 to 5, for 1, 4, 8, and 24 h, respectively. The extendedproducts were quantified by densitometric scanning of the autora-diogram, and the fold induction relative to that at time zero wasplotted versus the induction time.

observed (between 0 to 4 h), which presumably accounts forthe time needed to transcribe and translate the CAT mRNA.A maximum level of CAT protein was reached within 24 h,and in the continued presence of PMA, the enzyme levelsfell because transcription of the transfected gene was shut offdue to the absence of NF-KB.To confirm that activation of the gene was taking place at

a transcriptional level, we analyzed (by primer extension)cellular RNA prepared from KCAT2 cells stimulated forvarious periods of time (Fig. 3B). Correctly initiated RNAwas observed (inset, Fig. 3B), and quantitative densitometryshowed that the kinetics of transcription activation followedclosely the time course of factor induction. Thus, increasedtranscription was observed within 1 h poststimulation (Fig.3B, inset, lane 2; even though increased CAT activity wasnot observed till 4 h poststimulation), reached a maximum atapproximately 4 h (Fig. 3B, inset, lane 3), and was notdetectable after 24 h (Fig. 3B, inset, lane 5) of stimulation.There was a maximal eightfold level of induction of theRNA. This time course is very similar to that observed byImbra and Karin (15), who studied the induction of an SV40

enhancer-driven thymidine kinase (tk) gene in HepG2 cellsby phorbol esters. However, in that case the factors medi-ating the activation were not known, and therefore the timecourse of RNA induction could not be directly correlated tothe presence or absence of a specific factor. Recently, Chiuet al. (5) suggested that the PMA inducibility of the SV40enhancer arises from the combined action of three elementspresent within the enhancer (13), each of which is individu-ally PMA inducible. However, identification of the factorsthat interact with each of these sites has still not provided asatisfactory explanation for the observed kinetics of tran-scription activation. In our case, the experimental designsuggests that the activation is mediated by NF-KB, and thekinetics of RNA induction are entirely consistent with thetransient appearance of this factor under stimulatory condi-tions. To rule out the possibility that PMA was inducing amore general activation of cellular transcription, the endog-enous actin locus was analyzed by primer extension. It wasquite clear that over the course of the experiment, actinmRNA did not follow the pattern of expression seen foreither the CAT enzyme or cat RNA (data not shown). Thus,the effects observed were specific for the transfected geneand presumably due to an induced transcription factor thatcould interact with the K3 fragment-in all likelihood,NF-KB.

DISCUSSION

The K3 fragment of the K enhancer binds strongly only toa B-cell-specific factor, NF-KB. Our results show that mul-timerization of this sequence generates a powerful B-cell-specific regulatory element. Thus, a dimer (data not shown)or a trimer of this fragment can activate transcription of theSV40 early promoter by over 50-fold in plasma cells. Fur-thermore, this activity is restricted to the B-cell and plasmacell stages of B-cell differentiation only and is not observedin cells of the pre-B phenotype. The pattern of activitycorrelates precisely with the tissue distribution of the Kenhancer-binding factor NF-KB and strongly suggests thattranscription activation is dependent on NF-KB, the onlyprotein detected so far that binds to this DNA fragment. It isinteresting that the activity of the intact K enhancer inexactly the same context (location relative to the SV40promoter) was much lower (fourfold enhancement) than thatobtained by trimerizing one fragment of the enhancer. IfB-cell-specific enhancement can be mediated only byNF-KB, what then is the role of the various E motifs (3, 25,30, 35) of the K enhancer? We speculate that since NF-KB isinducible in other cell types (see below), this factor isutilized by nonlymphoid cells for transcription of other genesunder certain conditions. If the K gene were only underNF-KB control, perhaps it would also get transiently acti-vated in other cell types. The combination ofNF-KB with theE sites apparently confers the right degree of regulation thatmakes the K gene active only in B cells.Treatment of HeLa cells with active phorbol esters in-

duced a protein that bound to the K3 fragment and, on thebasis of in vitro competition experiments, appeared to beindistinguishable from NF-KB (31). Our results show thattranscription of a gene dependent on the K3 sequence canalso be induced in HeLa cells, suggesting that the inducedprotein is functionally similar to NF-KB as well. From theDNA-binding specificity and the activity, it appears that theinduced protein is in fact NF-KB itself. The kinetics oftranscription induction are also entirely consistent with thisidea. CAT enzyme levels reached a maximum approximately

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3530 NELSEN ET AL.

24 h after stimulation and then decreased. The decrease canbe explained by the fact that induction of the binding proteinwith PMA was also transient, disappearing within 8 h afterinduction (31). The kinetics also correlate well with theappearance of K light-chain protein when the cell line 70Z isstimulated with PMA (29). 70Z is a pre-B-cell line whichcontains one rearranged K allele and one unrearranged allelethat are normally transcriptionally silent. Treatment of 70Zcells with PMA induces K protein, and surface expression ofimmunoglobulin reaches a maximum after 24 h. This pre-sumably reflects the time required for K gene transcription tobe turned on and the protein made and transported to the cellsurface. As might be expected, the kinetics of cat mRNAfollowed the kinetics of NF-KB induction more closely andreached a maximum at about 4 h poststimulation.The time course of cat mRNA induction was very similar

to the time course of tk mRNA induction dependent on theSV40 enhancer sequences (15). It has been suggested thatthe PMA inducibility of the SV40 enhancer is mediated bythe transcription factor AP-3 (5). However, since AP-3binding activity is detectable in uninduced HeLa cells, thePMA-induced activation must proceed through currentlyunknown mechanisms. The striking similarity between thekinetics of transcription induction mentioned above and thesimilarity of the AP-3 and NF-KB binding sites suggest analternative mechanism for SV40 enhancer induction. Al-though AP-3 present in uninduced HeLa cells can interactwith the SV40 enhancer and may be required for constitutiveenhancer function, perhaps under stimulatory conditions it isthe induced NF-KB (as defined by its ability to interact withthe K3 DNA fragment) that interacts with the same sequenceelement to confer the property of inducibility. Such a mech-anism readily explains the induction time course observed asthat of NF-KB induction without having to invoke unknownmechanisms modulating the activity of AP-3. In the experi-ments described in this paper, we were able to discriminatebetween AP-3 activity and NF-KB activity by virtue of thefact that the foi-mer bound to the K3 DNA fragment veryweakly (if at all), whereas NF-KB bound to the fragmentstrongly (based on in vitro binding experiments that showedthat the K3 fragment does not form a nucleoprotein complexin uninduced HeLa cells extracts, whereas it does so inextracts from PMA-induced HeLa cells [31]). Recently, twoother transcription factors, AP-1 and AP-2, have been shownto interact with sequences required for PMA inducibility (2,14, 19, 20, 23). The K3 fragment does not appear to bindeither AP-1 or AP-2 (R. Tjian, personal communication),which is consistent with the tissue distribution and sequencespecificities of these factors and argues strongly against thepossibility that the effects described in this paper are beingmediated by these proteins.Tumor-promoting phorbol esters cause a wide variety of

effects (4, 32, 36), presumably by binding to and activatingcellular protein kinase C. Some of these effects are probablydue to the differential transcriptional regulation of endoge-nous genes. For example, the rat prolactin gene (8, 34), thehuman metallothioenin and collagenase genes (16), and theproenkephalin gene (7) are strongly stimulated by the actionof phorbol ester. The rapid induction of NF-KB and NF-KB-dependent transcription in HeLa cells after PMA treatmentclearly shows that a mechanism for tumor promotion mayinvolve alteration of cellular transcription by directly acti-vating transcription factors.

ACKNOWLEDGMENT

This work was supported by a grant from the National Institutesof Health (ALY 1 R29 GM38925-01).

ADDENDUM IN PROOF

Recently, Pierce et. al. (Proc. Natl. Acad. Sci. USA85:1482, 1988) have demonstrated transcriptional activationby the NF-KB-binding site in mitogen-stimulated T-lymphoidcells.

LITERATURE CITED1. Allesandrini, A., J. H. Pierce, D. Baltimore, and S. V. Desiderio.

1987. Continuing rearrangement of immunoglobulin and T-cellreceptor genes in a Ha-ras-transformed lymphoid progenitorcell line. Proc. Natl. Acad. Sci. USA 84:1799-1803.

2. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J.Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. 1987. Phorbolester-inducible genes contain a common cis element recognizedby a TPA-modulated trans-acting factor. Cell 49:729-739.

3. Augerau, P., and P. Chambon. 1986. The mouse immunoglobu-lin heavy chain enhancer: effect on transcription in vitro andbinding of proteins present in HeLa and lymphoid B cellextracts. EMBO J. 5:1791-1797.

4. Banerji, J., L. Olson, and W. Schaffner. 1983. A lymphocyte-specific cellular enhancer is located downstream of the joiningregion in immunoglobulin heavy chain genes. Cell 33:729-740.

5. Chiu, R., M. Imagawa, R. J. Imbra, J. R. Bockoven, and M.Karin. 1987. Multiple cis and trans-acting elements mediate thetranscriptional response to phorbol esters. Nature (London)329:648-651.

6. Citri, Y., J. Braun, and D. Baltimore. 1987. Elevated mycexpression and c-myc amplification in spontaneously occuringB-lymphoid cells lines. J. Exp. Med. 165:1188-1194.

7. Comb, M., N. C. Birnerg, A. Seasholtz, E. Herbert, and H. M.Goodman. 1986. A cyclic AMP and phorbol ester-inducibleDNA element. Nature (London) 323:353-356.

8. Elsholtz, H. P., H. J. Margalam, E. Potter, V. R. Albert, S.Supourt, R. M. Evans, and M. G. Rosenfeld. 1986. Two differentcis-active elements transfer the transcriptional effects of bothEGF and phorbol esters. Science 234:1552-1557.

9. Garcia, J. V., L. Bich-Thuy, J. Stafford, and C. Queen. 1986.Synergism between immunoglobulin enhancers and promoters.Nature (London) 322:383-385.

10. Gerster, T., P. Matthias, M. Thali, J. Jiricny, and W. Schaffner.1987. Cell type-specificity elements of immunoglobulin heavychain gene enhancer. EMBO J. 6:1323-1330.

11. Gillies, S. D., S. L. Morrison, V. T. Oi, and S. Tonegawa. 1983.A tissue-specific transcriptional enhancer element is located inthe major intron of a rearranged immunoglobulin heavy chaingene. Cell 33:717-728.

12. Grosschedl, R., and D. Baltimore. 1985. Cell-type specificity ofimmunoglobulin gene expression is regulated by at least threedifferent DNA sequence elements. Cell 41:885-897.

13. Herr, W., and G. Clarke. 1986. The SV40 enhancer is composedof multiple functional elements that can compensate for oneanother. Cell 45:461-470.

14. Imagawa, M., R. Chiu, and M. Karin. 1987. Transcription factorAP-2 mediates induction by two different signal transductionpathways: protein kinase C and cAMP. Cell 51:251-260.

15. Imbra, R., and M. Karin. 1986. Phorbol ester induces thetranscriptional stimulatory activity of the SV40 promoter. Na-ture (London) 323:555-558.

16. Imbra, R., and M. Karin. 1987. Metallothionein gene expressionis regulated by serum factors and activators of protein kinase C.Mol. Cell. Biol. 7:1358-1363.

17. Kadesch, T., P. Zerros, and D. Ruezinsky. 1986. Functionalanalysis of the murine IgH enhancer: evidence for negativecontrol of cell-type specificity. Nucleic Acids Res. 14:8209-8221.

18. Landolfi, N. F., J. D. Capra, and P. W. Tucker. 1986. Interac-tion of cell-type-specific nuclear proteins with immunoglobulin

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VH promoter region sequences. Nature (London) 323:548-551.19. Lee, W., A. Haslinger, M. Karin, and R. Tjian. 1987. Activation

of transcription by two factors that bind promoter and enhancersequences of the human metallothionein gene and SV40. Nature(London) 325:368-372.

20. Lee, W., P. Mitchell, and R. Tjian. 1987. Purified transcriptionfactor AP-1 interacts with TPA-inducible enhancer elements.Cell 49:741-752.

21. Lenardo, M., J. W. Pierce, and D. Baltimore. 1987. Proteinbinding sites in immunoglobulin gene enhancers determinetranscription rates and inducibility. Science 236:1573-1577.

22. Mercola, M., X. F. Wang, J. Olsen, and K. Calame. 1983.Transcriptional enhancer elements in the mouse immunoglobu-lin heavy chain locus. Science 221:663-665.

23. Mitchell, P., C. Wang, and R. Tjian. 1987. Positive and negativeregulation of transcription in vitro: enhancer-binding proteinAP-2 is inhibited by SV40 T antigen. Cell 50:847-861.

24. Neuberger, M. S. 1983. Expression and regulation of immuno-globulin heavy chain gene transfected into lymphoid cells.EMBO J. 2:1373-1378.

25. Peterson, C. L., K. Orth, and K. L. Calame. 1986. Binding invitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA. Mol. Cell. Biol. 6:4168-4178.

26. Picard, D., and W. Schaffner. 1984. A lymphocyte specificenhancer in the mouse immunoglobulin kappa gene. Nature(London) 307:80-82.

27. Queen, C., and D. Baltimore. 1983. Immunoglobulin gene tran-scription is activated by downstream sequence elements. Cell33:741-748.

28. Queen, C., and J. Stafford. 1984. Fine mapping of an immuno-

globulin gene activator. Mol. Cell. Biol. 4:1042-1049.29. Rosoff, P. M., and L. C. Cantley. 1985. Lipopolysaccharide and

phorbol esters induce differentiation but have opposite effectson phosphatidylinositol turnover and Ca"+ mobilization in 70Z/3 pre-B lymphocytes. J. Biol. Chem. 260:9209-9215.

30. Sen, R., and D. Baltimore. 1986. Multiple nuclear factorsinteract with the immunqglobulin enhancer sequences. Cell 46:705-716.

31. Sen, R., and D. Baltimore. 1986. Inducibility of K immunoglob-ulin enhancer-binding protein NF-KB by a posttranslationalmechanism. Cell 47:921-928.

32. Slaga, T. J. 1983. Cellular and molecular mechanisms of tumorpromotion. Cancer Surveys 2:595-612.

33. Staudt, L. M., H. Singh, R. Sen, T. Wirth, P. A. Sharp, and D.Baltimore. 1986. A lymphoid-specific protein binding to theoctamer motif of immunoglobulin genes. Nature (London) 323:640-643.

34. Supowit, S. C., E. Potter, R. M. Evans, and M. G. Rosenfeld.1984. Polypeptide hormone regulation of gene transcription:specific 5' genomic sequences are required for epidermal growthfactor and phorbol ester regulation of prolactin gene expression.Proc. Nati. Acad. Sci. USA 81:2975-2979.

35. Weinberger, J., D. Baltimore, and P. A. Sharp. 1986. Distinctfactors bind to the homologous sequences in the immunoglob-ulin heavy chain enhancer. Nature (London) 322:846-848.

36. Weinstein, I. B., L. S. Lee, P. B. Fisher, A. Mufson, and H.Yamasaki. 1979. Action of phorbol esters in cell culture: mim-icry of transformation, altered differentiation and effects on cellmembrane. J. Supramol. Struct. 12:195-208.

VOL. 8, 1988