Vol. 4, 753-760, September 1993 Cell Growth & Differentiation 753

Serum Deprivation Response Gene Is Induced by SerumStarvation but not by Contact Inhibition’

S. Gustincich2 and C. Schneider

International Center for Genetic Engineering and Biotechnology and

Laboratorio Nazionale, Consorzio lnteruniversitario Biotecnologie, AreaScience Park, Padriciano 99, 3401 2 Trieste, Italy

Abstrad

The relationship between quiescence induced by serumdeprivation and that induced by high cell density(contad inhibition) is still unclear. Here, we describe agene, named sdr (serum deprivation response), whosemRNA level is highly induced in serum starved but notin density dependent growth arrested NIH3T3 cells. sdrindudion seems to be coupled to growth arrest sinceit does not become apparent when transformed NIH3T3cells are cultured in low serum. The expression of sdris down-regulated within 6 h after the addition of serumor epidermal growth fador to serum starved cells.Although a transient reindudion is noticed at later timesafter fetal calf serum stimulation, this is not the case forepidermal growth fador, for which the sdr mRNA levelremains down-regulated. The data presented herepoint to the possibility that the density dependentgrowth arrest state cannot be simply explained by localgrowth fador depletion, as occurs in overcrowdedcultures, but it could be the consequence of a morecomplex pathway mediated by cellular interadions.

IntrodudionAlthough growth factor availability allows cells to undergoa series ofcell divisions (“in cycle” state), serum deprivationor high cell density (contact inhibition) restricts their growthinto an “out of cycle” state referred to as C0 (i-5).

Pioneering studies have focused on the basic biochemicalproperties of quiescent cells such as smaller cell size, de-creased macromolecular synthesis, and monosomal struc-tune of nibosomes (6). More recently, RNAs and proteins thatare specifically present in growth arrested cells have beenidentified in different systems (7-u). By subtractive hybrid-ization screening of a G0 enriched cDNA3 library, six genesspecifically expressed in quiescent NIH3T3 cells werecloned: these genes were named gas (growth arrest specific)genes (i 2-i 6). They are induced by both serum starvation

Received 1/22/93; revised 6/1 8/93; accepted 6/30/93.

1 This work was supported by funds from the Associazione Italiana per

Ia Ricerca sul Cancro and the Consiglio Nazionale delle Ricerche-ProgettoFinalizzato Applicazioni Cliniche Ricerche Oncologiche to C. S.2 Present address: Department of Neurobiology, Harvard Medical School,220 Longwood Avenue, Boston, MA 0211 5. To whom requests for reprints

should be addressed.3 The abbreviations used are: cDNA, complementary DNA; EGF, epidermalgrowth factor; poIy(A)�, polyadenylated; FCS, fetal calf serum; gapdh,glyceraldehyde-3-phosphate dehydrogenase; BrdUrd, bromodeoxyuridine;aa, amino acid; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate;SSC, standard saline citrate.

and contact inhibition. However, the relationship betweenquiescence induced by serum starvation and that induced bycontact inhibition is still unclear.

The phenomenon ofcontact inhibition was first describedas cessation ofcell movement when two adjacent cells comeinto contact; by extension, this concept was then applied tothe consequent inhibition of cell division (i 7-i9). Sincethen, a debate has focused on the relevance of cell to cellcontact for the establishment of the quiescent state.

It has been proposed that growth inhibition is mainly dueto growth factor deprivation rather than cell to cell interac-tion. In accordance with this, even the increased growthnoticed at the margin of a wound in a confluent monolayerhas been explained as a local increase of growth factor con-centration (20, 21).

All of the data accumulated so far have not been able todissect the tight relationship between such growth arreststates. Recently, however, a specific increase in tyrosinephosphatase activity has been reported in density dependentgrowth arrested cells which seems to be present at a lowerlevel in growing or serum deprived Swiss 3T3 cells (22). Wethus decided to investigate the possible existence of differentgene expression patterns in the two quiescent states.

This report describes the cloning and analysis of a genethat is highly expressed during the out ofcycle state inducedby serum starvation but not by contact inhibition. Interest-ingly, this new gene is also expressed, albeit at a lower level,in growing cells. In addition, although showing no suchregulation when transformed cells are cultured in low serum,sdr expression is completely shut off after EGF growth in-duction in nontransformed cells. The machinery involved inits coordinate regulation is here examined and discussed.

Results

Cloning of Genes Highly Induced in Serum Deprived Cellsbut not in Serum Stimulated and Density DependentGrowth Inhibited Cells. An oriented cDNA library was pre-pared from the poly(AY” RNAfraction isolated from NIH3T3cells starved for 48 h in 0.5% FCS. Under these conditions,less than 5% of cells were in S phase.

In order to clone genes that are specifically related to the

growth arrest state induced by serum deprivation, we de-cided to screen a G0 serum starved cDNA library with threecDNA probes synthesized from: (a) resting NIH3T3 cells cul-tuned for 48 h in low serum; (b) serum stimulated cells at 6h after addition of 20% FCS to starved cells; (c) density de-pendent growth inhibited cells that had been cultured for 8days with the medium replaced every 2 days. This differ-ential hybridization screening is obviously restricted to theanalysis of the high abundance class of transcripts.

Clones that are highly expressed in serum starved but notin serum stimulated and density dependent growth arrestedcells were purified, rescneened with the respective cDNAprobes, and further analyzed.

After restriction and cross-hybridization analysis, 35plaques appeared to be cDNAs of different lengths of the

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Fig. 1. Analysis of sdr mRNA levelduring growth arrest induction.

4- sdr Northern blot analysis was per-formed on equal amounts of totalRNA(i 0 hg) isolated from (A) grow-ing NIH3T3 cells and, at the mdi-

cated times after 0.5% FCS addition,(B) actively growing cells that were

4-. gapdh kept thereafter in the same dish with1 0% FCS for different times (culture

medium containing 10% FCS wasreplaced every 2 days). The sameblot was probed, as indicated, withsdr and gapdh cDNAs. The analysisof DNA synthesis levels for eachtime course is shown at bottom.I0

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same mRNA species, with a relative abundance in the Ii-brary of 0.1 3%. The longest clone was 3 kilobases long; itwas named sdr (serum deprivation response) and furthercharacterized.

Regulation of sdr Expression after Serum Starvation andDensity Dependent Growth Inhibition. The level of sdrmRNA expression was analyzed on Northern blots by usingtotal RNA extracted at various times after serum deprivationofexponentially growing NIH3T3 cells. Cells were shifted to0.5% FCS 24 h after seeding in i 0% FCS. sdr probe recog-nizes a 3-kilobase-long transcript that is normally expressedat a relatively low level in growing cells and then increasesduningthe initial 1 2 h of0.5% serum addition. Later, it beginsto decrease at 1 8 h, reaching a minimal level at 24 and 36h. Then the mRNA is highly induced after 48 h (Fig. iA).

Normalization of RNA amounts was performed by prob-ing the same Northern blot with gapdh.

The percentage of cells in S phase was monitored by ana-lyzing BrdUrd incorporation in cells grown on covenslips foreach time point. Less than 1 5% of the cells remained in Sphase 24 h after serum starvation.

To assess the expression of sdr mRNA during density de-pendent growth inhibition, NIH3T3 cells were seeded ini 0% FCS, and fresh medium containing 1 0% FCS was re-placed every 2 days. The sdr mRNA level in growing cells

decreases as soon as 2 days after seeding and is maintainedat a lower but detectable level during the complete timecourse (Fig. 1 B).

gapdh control expression level did not change, and DNAsynthesis analysis showed a significant decrease as early as2 days after seeding.

Regulation of sdr Expression in Transformed Cell Lines.To examine whether sdr mRNA induction in low serum istightly related to the growth state of the cells or is broughtabout by a general stress response due to lack of growthfactors, we analyzed sdr expression in a panel of NIH3T3transformed cell lines cultured in 0.5% FCS (1 5, 1 6). Fig. 2

shows that whereas sdr mRNA is highly expressed in un-transformed fibroblasts after 48 h in low serum, under thesame conditions, v-myc, v-fos, v-ras, and v-src NIH3T3transformed cell lines do not present any induction ofthe sdrgene. Similar results were obtained using SV4O transformedNIH3T3 cells (data not shown).

The amount of RNA loaded on each gel was evaluated byhybridization with the gapdh probe on the same Northernblot.

In order to analyze the proliferation pattern of each cellline used for Northern analysis, S phase assay using a 2-hlabeling period with BrdUnd was performed on cells grownon coverslips in the same culture dish from which RNA wasextracted. The histogram shows that although nontrans-formed NIH3T3 cell cultures present a very low percentageof cells in S phase, all of the transformed cell lines are stillcycling after 48 h in low serum.

Regulation of sdr Expression during G0-’S Transition. Atthis point, we wanted to analyze the kinetics of sdr down-regulation when serum deprived NIH3T3 cells are stimu-lated to reenter the cell cycle. Fig. 3 shows the expressionof sdrmRNA at different times after a synchronous inductionof the cell cycle with 20% FCS in NIH3T3 cells that werepreviously arrested for 48 h in 0.5% FCS. The mRNA iden-tified by sdr probe is abundantly expressed in arrested cells(time 0) and has the lowest expression 6 h after addition ofserum. Surprisingly, the mRNA transiently increases 12 hafter serum addition.

Normalization of RNA amounts was performed with thegapdh probe on the same blot, and the percentage of cellsin S phase was measured by analyzing BrdUrd incorporationin cells growing on covenslips for each time point after serumaddition.

Given the complexity of sdrexpression during a synchno-nous cell cycle induction as driven by serum, we wanted toanalyze whether any single mitogenic growth factor is ca-pable of inducing a simpler pattern of sdr down-regulation.

Fig. 3. Kinetics of sdr mRNA expression during -‘,transition. NIH3T3cells were arrested for 48 h in 0.5% FCS and stimulated to reenter the cellcycle with the addition of 20% FCS. After the indicated intervals, RNA wasisolated and analyzed by Northern blot (10 pg of total RNA( with sdr andgapdh probes. The percentage of cells in S phase is shown at bottom of each

time course.

Cell Growth & Differentiation 755

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transformed (a) and v-los (b), v-myc (c(, v-ras (d(, and v-src (e) transformedNIH3T3 cells were cultured for 48 h in 0.5% FCS. RNA was isolated and

analyzed by Northern blot (1 0 pg of total RNA) with sdr and gapdh probes.The percentage of cells in S phase is shown at bottom.

Among the various growth factors tested, ECF was the onlyone showing a clean-cut response during C0-sS transition.As can be seen in Fig. 4, ECF is able to down-regulate sdrrnRNA expression within the first 6 h after addition to serumstarved NIH3T3 cells. Most importantly, this growth factorseems to completely abolish sdr reinduction during C1-Stransition as observed with serum.

Normalization of RNA amounts was performed with thegapdh probe on the same blot. The histogram shows the

percentage of cells in S phase as measured by BrdUrd in-corporation in cells growing on coverslips for each timepoint after ECF addition; it can be seen that ECF is able toinduce cycle reentry as efficiently as FCS.

Posttranscriptional Regulation of sdr Expression duringG0-.S Transition. To better understand the molecularmechanisms involved in the control of sdrexpression duringcycle reentry as elicited by FCS, we first performed a run-onexperiment on nuclei collected at the same times after FCSaddition to C0 NIH3T3 cells as analyzed for steady-statemRNA level. As controls for sdr transcriptional level, we

used gapdh, known to remain constant, and gas!, whosetranscription is strongly down-regulated 6 h after addition ofserum (23). Fig. 5 showsthe resultsofsuch an analysis. It canbe assessed that sdr transcriptional level does not seem toappreciably change during the time points analyzed in thenuclear run-on, whereas the controls show the expected pat-terns. These results strongly suggest that a posttranscniptionalmechanism is responsible for sdr mRNA regulation.

We thus analyzed sdr mRNA steady-state levels in serumdeprived C0 NIH3T3 cells either alone or in the presence ofactinomycin D or cycloheximide as generally used to blockfurther RNA transcription or protein synthesis, respectively.

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Fig. 6A shows that, under growth arrest conditions, sdrmRNA half-life is at least4 h long and is not affected by eitheractinornycin D or cycloheximide.

Since down-regulation of sdr rnRNA steady-state level has

been demonstrated for FCS and ECF with different patterns,we tried to dissect its dependency on de novo RNA andprotein biosynthesis.

Fig. 6B shows that, in the case of serum stimulation, theconcomitant addition of actinornycin D and cyclohexirnideinhibits the down-regulation of sdr rnRNA during C0-”C1transition. Thus, both RNA and protein synthesis seem to berequired for the down-regulation of sdr rnRNA during the

early serum response.Fig. 6Cshows that, in the case of ECF addition, treatment

with actinomycin D does not completely inhibit sdr rnRNA

down-regulation, in contrast with cycloheximide, which isstill able to block it. Thus, de novo transcription seems notfully required for sdr mRNA down-regulation induced byECF, whereas active protein biosynthesis is still required in

both ECF and FCS induced sdr down-regulation.Expression of sdr mRNA in Various Tissues. In order to

analyze the steady-state level of sdr mRNA present in vivo,total RNA was extracted from various mouse tissues. ByNorthern blot analysis, sdr is seen to be highly expressed inlung; it is also highly expressed in heart and kidney. The

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Fig. 5. Nuclear run-on analysis of sdrtranscription during the growth cycle.Nuclei were isolated from NIH3T3 cells at growth arrest (48 h in 0.5”/o FCS)

and at various times after 20% FCS addition. The nuclear preparations wereallowed to incorporate (a-#{176}PIUTP, and the recovered 2P labeled RNA was

allowed to hybridize to denatured and immobilized plasmid DNA containingthe indicated cDNA inserts.

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Fig. 7. Expression of sdr mRNA in mouse tissues. Total RNA was extractedfrom murine organs and analyzed on Northern blot (20 pg) using sdr cDNA

as probe. Tissues indicated are as follows: Lu, lung; He, heart; Ut, uterus; Th,

thymus; In, intestine; Sp, spleen; Ki, kidney; St, stomach; Li, liver. 3T3,NIH3T3 cells for 48 h in 0.5% FCS.

756 Regulation of sdr Expression

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Fig. 4. Regulation of sdr mRNA by EGF. NIH3T3 cells were arrested for 48

h in 0.5”/o FCS and stimulated to reenter the cell cycle with the addition ofserum-free medium containing 40 ng/ml of EGF. After the indicated intervals,

RNA was isolated and analyzed by Northern blot (10 pg of total RNA) withsdr and gapdh probes. The percentage of cells in S phase is shown at bottomof each time course.

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oftotal RNA (10 pg) extracted from (A) 48 h in 0.5% FCS and at the indicatedtimes in the absence (-) or presence (+) of actinomycin D or cycloheximideorfrom (B) 20#{176}kFCS or (C) EGF activated cells in the presence of actinomycin

D or cycloheximide.

other analyzed tissues (liven, spleen, thymus, stomach, in-testine, and uterus) present a detectable level of sdr expres-sion. The size of sdr mRNA is apparently similar in all of thetissues analyzed, as observed in NIH3T3 cells (Fig. 7).

sdrcDNASequence. Thecompletesequenceof the long-

est sdr cDNA clone comprises 2909 nucleotides (Fig. 8A).

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The 5’ untranslated region is 118 nucleotides long andcontains stop codons in all three open reading frames. Alarge open reading frame of 1 254 nucleotides encodes fora protein of 4i8 amino acids with a predicted molecularweight of 46,600. The ATG at position ii 9 resemblesKozak’s consensus sequences for the initiation of transla-tion (24). The 3’ untranslated region, showing frequentstop codons in all three frames, is i537 nucleotides long.

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100 110 120 130 140

Cell Growth & Differentiation 757

Fig. 8. A, DNA and deduced pro-

tein sequence of the cDNA clone of

sdr gene. The 3’ untranslated se-quence shows two putative instabil-ity motifs, ATTTA (underlined) and a

canonical consensus signal ATTAAAfor poly(A)’ addition at position2878. 8, amino acid comparison be-

tween the deduced sdr protein and ahuman cellular protein fused to ac-tivated c-raf (clone 7N-i). Compari-son was performed by using analignment program against Swiss

Prot protein sequence data bank.

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Identity - 49.5%Similarity - 58.5%

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758 Regulation of sdr Expression

4 5, Gustincich et al., manuscript in preparation.

It contains two consensus elements (AUUUA) involved inmRNA stability (25) and a canonical polyadenylation sig-nal, AUUAAA, at -32 nucleotides (2878) (26). The pre-dicted sdr amino acid sequence was compared with thecurrent protein database releases and DNA sequences us-ing the Intelligenetics software package. The region fromaa 53 to aa 1 75 shows 49.5% identity and 58.5% similar-ity with the sequence from aa 24 to aa 1 46 of the human7N-i clone, which is an activated form of the c-raf gene(Fig. 8B) (27). The first 568 amino acids at the NH2 tenmi-nus of this oncogenic fusion protein are formed by a pre-viously undescnibed human cellular gene. This homologycould suggest that sdr is a member of a new gene family.Other interesting similarities with neurofilaments (13%),microtubule associated protein iB, and panamyosin that

appear in the a-helical-rich domains may be the result ofthe presence of common secondary structures.

Discussion

Two methods are commonly used for establishing growtharrest in vitro: growth factor deprivation and contact inhi-bition (1 , 4). It is still an open question whether these twogrowth arrest states are achieved and/or maintained through

different mechanisms that rely on different gene expressionprograms (1 2, 1 7-i 9, 21). We decided to approach thisquestion by cloning genes that are differentially induced byserum deprivation but not by contact inhibition in NIH3T3cells.

Use of the differential hybridization screening protocolresulted in the selection of clones corresponding to theabundantly expressed class of transcripts: all of the positiveclones were, in fact, copies of the same mRNA species. Wenamed this gene sdr.

The induction of sdr mRNA by serum deprivation is quitecomplex (Fig. i): a first induction of sdr mRNA (at 1 2 h) isfollowed by a transient decrease (24-36 h) before reachingthe final high level of expression (48 h).

However, cells arrested by density dependent inhibitionalways maintain sdr expression at a very low level evenwhen growth arrest is fully established.

The same sdr induction noted in NIH3T3 cells has beenalso proved similar when IMR9O human fibroblasts weregrowth arrested by either low serum on density dependentinhibition.4

To assess whether sdr induction by low serum reflectsthe existence of a specific cellular response related to se-rum deprivation on whether it is the result of experiencingstress conditions, we analyzed sdr mRNA levels in trans-formed cell lines similarly grown under low serum condi-tions. All of the various single oncogene transformedNIH3T3 cells tested seem to have lost the growth factor re-quirement for proliferation, as shown by the high percent-age of cells remaining in S phase after 48 h of low serumculture. Under these conditions, in all cases there is noappreciable induction of sdr mRNA, thus suggesting a pos-sible coupling between cell proliferation control and sdrexpression (Fig. 2).

We then analyzed the kinetics of sdr down-regulationwhen growth arrested cells are synchronously induced toenter the growth cycle by addition of serum or variousgrowth factors. A complex pattern of sdr expression was

noted after addition of serum, which includes an initialdown-regulation ofsdrat 6 h followed by a transient increaseat 1 2 h after serum addition (Fig. 3); this pattern was alsoconfirmed in the IMR9O cells.4

Given this complex pattern of regulation of sdr expres-sion during serum induction of growth arrested cells, wetested whether any single mitogenic growth factor is ca-pable of completely repressing sdr expression. Althoughplatelet derived growth factor, fibroblast growth factor, in-sulin, and 4�3-phorboI 1 2-mynistate 1 3ct-acetate were ableto down-regulate sdr 6 h after their addition, a further in-crease of sdr was always observed (data not shown). Theonly growth factor that is able to completely shut off sdrexpression 6 h after its addition was shown to be EGF (Fig.4) (28-34).

We thus analyzed the mechanisms that could be nespon-sible for such different patterns of sdr regulation duringG0-sS transition. From the nuclear run-on analysis, wecould first exclude a clean involvement of transcriptionalregulation (Fig. 5). By the use of drugs such as actinomycin

D or cycloheximide, we could determine that active proteinbiosynthesis seems to be required both for EGF and FCS sdrdown-regulation during G0-sG1 transition (Fig. 6). Thiscould suggest either an involvement ofthe primary responsegenes in sdrdown-regulation on a feedback control executedby the sdr protein product on the sdr mRNA level itself.Moreover, de novo RNA biosynthesis seems to be fully re-quired only for serum mediated sdrdown-regulation and notfor the EGF effects.

These results could be explained either as a dosageeffect of EGF addition compared to its amount presentin 20% FCS, on by the presence in the serum of addi-tional components able to act on sdr mRNA level, possiblyrelated to different pathways used by different mitogenicsignals (33, 34).

According to this last hypothesis, it has been reported thatantisense oligodeoxynucleotides against casein kinase II caninhibit the EGF but not the serum induction of DNA nepli-

cation in human fibnoblasts (35).Furthermore, gene expression patterns specifically in-

duced by EGF have recently been elucidated through thestudy of genes that are induced in quiescent rat fibroblastsby reactivation of temperature sensitive v-src and v-fpsmutants (36).

The deduced protein sequence of sdr has not helped inthe understanding of the biological function of this gene.Its partial homology with an activated form of c-raf couldprobably reflect the existence of protein domains relatedto sdr (27).

Together with the expression of common growth arrestspecific genes (1 2, 1 3) that define the out of cycle state, theexistence ofgenes like sdrthat are differentially expressed inserum deprived and contact inhibited quiescent cells couldsuggest that the density dependent growth arrest state is notsimply explainable by a local growth factor depletion in anovercrowded culture but seems to imply a more complexresponse. This difference is probably related to the relative“growth restrictions” imposed: in the former, the presence oflow amounts of growth factors induces the expression ofgenes that will be more sensitive to the presence or absenceof different mitogens; in the latter, cell to cell and cell toextracellulan matrix interactions/communications are themost relevant restrictions even in the presence of highamounts of growth factors.

Cell Growth & Differentiation 759

2. Zetterberg, A., and Larsson, 0. Kinetic analysis of regulatory events in G,

Materials and Methods

Cell Lines and Cell Culture Conditions. NIH3T3 cells werekindly provided by Dr. R. Muller. They were routinely cul-tuned in Dulbecco’s modified Eagle’s medium with i 0%FCS, penicillin, and streptomycin (100 units/pg/mI).

For serum starvation, the medium was changed to 0.5%FCS when cells were subconfluent; cells were then left in thesame medium for 48 h. Under these conditions, labelingwith 50 �M BndUrd for an additional 24 h resulted in labelingof less than 1 0% of the nuclei.

For DNA synthesis induction, fresh medium containing10% or 20% FCS was added to the arrested cells. Cells werethen harvested at the desired times for RNA isolation. After24 h of BndUnd incorporation, more than 90% of the nucleiscored positive.

For density dependent inhibition, cells were plated at 10�/cm2 in 1 0% FCS. Beginning 24 h after plating (consideredas the starting point for growing cells), the medium waschanged every 2 days. After 4 days in culture, less than 5%ofthe cells were able to incorporate BrdUnd when incubatedfor 2 h.

In some experiments, entry into DNA synthesis wasinduced by addition of EGF (gently provided by A. Ulnich,Max Planck Institute, Munich, Germany) at a concentrationof 40 np/mI.

When used, cycloheximide and actinomycin D concen-trations were, respectively, 1 0 and 5 pg/mI. Cycloheximidewas able to inhibit more than 95% of protein synthesis asdetermined by [35S]methionine incorporation and trichlo-roacetic acid precipitation.

NIH3T3 cells transformed with v-myc, v-ras, and v-srcwere kindly provided by F. Tat#{244}(University of Rome, Rome,Italy). NIH3T3 cells transformed with v-foswene provided byR. Muller (University of Marbung, Manbung, Germany).NIH3T3 cells transformed with SV4O large T antigenwere provided by R. Martin (National Institutes of Health,Bethesda, MD) (1 5, 16).

DNA Synthesis Assay. Cells grown on covenslips (20mm x 20 mm), in the same culture dishes from whichRNA was prepared, were incubated for the described timein the presence of 50 �M BndUrd (Amensham Intenna-tional). After this time, cells were fixed (5 mm in methanolat 4#{176}Cfollowed by 5 mm in acetone at 4#{176}C).DNA wasthen denatured by treatment with 1 .5 N HCI for 1 0 mm.The covenslips, after washing with PBS, were incubatedwith mouse monoclonal antibody against BrdUrd for 1 hat 37#{176}C,washed twice in PBS, and then incubated for 45mm at 37#{176}Cwith tetramethylrhodammne isothiocyanateconjugated rabbit antimouse immunoglobulin antibodies(37). Total nuclei were visualized with Hoechst 33342stain (1 pg/mI). More than 300 nuclei were observed foreach covenslip. The percentage of activation was calcu-lated as the ratio between nuclei positive for tetnamethyl-rhodamine isothiocyanate and total nuclei (Hoechst).

Total RNA and PoIy(A)� Preparation. Total RNA was ex-tracted from cultured cells or animal tissues as previouslydescribed (38). The munine organs were conserved at -80#{176}C.The poly(A)’� fraction of total RNA was prepared using anoligo(dT) cellulose (Stratagene, San Diego, CA).

Construdion and Screening of a cDNA Library. ThecDNA library was constructed using 3 pg of poly(A) mRNAextracted from quiescent cells starved for 48 h in 0.5% FCS.First strand cDNA synthesis was performed using an oligo-(dT) primer as previously described (39). cDNA was ligatedto EcoRI/HindIlI linkers and cloned in an oriented way (40)

into the A vector T7-T3/E-H (41). The library contained 2 X105 recombinants.

For screening, 5 X 10” plaques of the amplified librarywere plated at a density of 5000 plaque forming units/1 50-mm Petni dish. Three lifts were made from each of i 0Petni dishes using nylon membranes (Gene Screen). The firstlift from each plate was probed with a hot single strandcDNA from serum starved cells; the second lift was probedwith cDNA from contact inhibited cells; and the third onewas probed with cDNA from cells harvested 6 h after theaddition of 20% FCS to growth arrested cells. Plaques thatspecifically hybridized only with the cDNA probe from se-rum starved cells were isolated and rescreened.

Northern Blot Analysis. Total cellular RNA (i 0 pg) wasfractionated on i % aganose gel containing 6.7% formalde-hyde. Integrity and relative amount of RNA were analyzedby ethidium bromide staining. Gels were transferred to aDunalon-UV nylon membrane (Stnatagene). RNA was cross-linked by exposure to UV light (Stnatalinker; Stratagene).Probe was prepared by random primer oligolabeling, andhybridization was performed in 1 M NaCI-i% SDS at 65#{176}Cusing 5 X 10� cpm/ml. Washes were performed as follows:2X SSC at room temperature twice for 5 mm each; 2X SSC-0.1 % SDS at 65#{176}Ctwice for 1 5 mm each.

DNA Sequencing. DNA fragments were sequenced di-nectly in A T7-T3/E-H vector as previously described (42)using the T7 DNA polymerase (Pharmacia). Synthetic oh-gonucleotides were also used as primers for the sequencingreactions. The entire sequence was read on both strands.

Run-on Experiment. Forthe isolation ofnuclei, cells werewashed twice with ice-cold PBS and removed with a rubberpoliceman. The pellet of cells was incubated for 5 mm inlysis buffer (10 m� Tnis-HCI, pH 7.4-10 mt�i NaCI-3 mistMgCl2-0.5% Nonidet P-40). The lysate was centrifuged at500 X g, and the pellet was resuspended in the storage buffer(10 mist Tnis-HCI, pH 8.3-40% glycenol-5 mist MgCI2-0.i misiEDTA, pH 8) and left in liquid N2 until use.

Run-on transcription assay was performed as previouslydescribed (43).

The DNA was spotted onto a nylon membrane (Strat-agene); i 0 pg of previously denatured DNA (0.25 M NaOHfor 20 mm at room temperature, neutralized by addition ofan equal volume of 0.25X SSC) was applied per slot. Hy-bnidization was performed in 1 M NaCI-i% SDS-i mg/mIhepanin-i 00 pg/mI salmon sperm DNA at 65#{176}Cfor 36 h witha probe concentration of 2 X i 06 cpm/ml. Filters were thenwashed with 2X SSC at room temperature twice for 10 mmeach, with 2X SSC-i% SDS at 65#{176}Cfor 15 mm, and finallywith 0.2x SSC-0.i% SDS at room temperature for iO mm.

Acknowledgments

We are grateful to Dr. A. Ulrich (Max Planck Institute, Munich, Germany) forproviding EGF and to Dr. F. Tatb (La Sapienza University, Rome, Italy) for

providing v-myc, v-ras, and v-srctransformed cell lines and for critically read-ingthe manuscript. NIH3T3 cellstransformed with v-foswere kindly providedby R. Muller (University of Marburg, Marburg, Germany), and NIH3T3 cellstransformed with SV4O large I antigen were kindly provided by R. Martin(National Institutes of Health, Bethesda, MD). We thank Drs. G. Manfioletti,

G. Del Sal, E. Ruaro, C. Brancolini, and S. Goruppi for their helpful sugges-tions and advice in performing some experiments. We also thank OriettaPoles, Stefania Marzinotto, and Steve Bottega for their technical assistance.

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