Overexpression of c-Myc Inhibits p21WAF1/CIP1 Expression andInduces S-Phase Entry in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-sensitive Human Cancer Cells1

Kyran O. Mitchell and Wafik S. El-Deiry2

Laboratory of Molecular Oncology and Cell Cycle Regulation, HowardHughes Medical Institute, Department of Medicine, Genetics, CancerCenter, and Institute for Human Gene Therapy, University ofPennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

AbstractThe c-Myc oncoprotein is a transcription factorinvolved in cellular transformation. We previously found(M. V. Blagosklonny, et al., Cancer Res., 57: 320–325,1997) that exposure of human SkBr3 breast cancer andLNCaP prostate cancer cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a growtharrest associated with the up-regulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and theinhibition of c-Myc expression. We show here thatexogenous c-Myc inhibits p21 expression in SkBr3 andLNCaP cells induced to enter into S-phase. p27expression was not increased from basal levels in TPA-treated growth–arrested cells. A time course afterinfection of TPA-arrested cells using a c-Myc-expressing adenovirus revealed that the inhibition ofp21 expression preceded entry into S-phase. Incontrast, after infection by E2F-1-expressingadenovirus, p21 expression was reduced after the cellsentered S-phase. Overexpression of c-Myc reduced thelevels of endogenous p21 mRNA, and transfection ofc-Myc repressed p21-promoter luciferase-reportergene expression. The results suggest that the down-regulation of p21 expression may contribute to c-Myc-dependent entry into S-phase, possibly in situations inwhich growth arrest is associated with increased p21expression.

IntroductionThe c-myc proto-oncogene has been found to be involved inthe progression of a wide range of neoplasias. c-Myc proteinhas been shown to act as a transcription factor in conjunc-tion with its transcriptional activation partner Max (1). c-Mycforms a heterodimer with Max and binds to the core hex-

anucleotide CACGTG (the “E” box; Ref. 2). This specific DNAbinding is mediated by the basic helix-loop-helix leucinezipper domain found in the COOH-terminal end of c-Myc. Inits NH2-terminal region, the c-Myc protein contains a trans-activation domain (3). Only a few transcriptional targets to themyc;Max heterodimer have been identified, including theadenovirus major-late promoter (4), eIF4E (5), car-bamoylphosphate synthase (cad) (6), a-prothymosin (7), or-nithine decarboxylase (ODC) (8), MrDb (9), ECA39 (10), andcdc25A (11). c-Myc may also act as a transcriptional repres-sor of C/EBPa (12, 4), cyclin D1 (13), the adenovirus 5 major-late promoter (4), thrombospondin-1 (14), and gadd45 (15).The mechanism of repression by c-Myc remains unclear.

We recently reported (16) that the down-regulation of c-myc expression may be a required late step in growth arrestfollowing phorbol ester TPA3 exposure of (TPA-sensitive)epithelial cancer cells. The growth arrest of the TPA-sensitiveSkBr3 human breast cancer and LNCaP human prostatecancer cells was associated with the induction of expressionof the cell cycle inhibitor p21WAF1/CIP1. p21 inhibits cell cycleprogression by binding cyclin-cyclin dependent kinases andinhibiting their kinase activity (17) as well as by binding to theproliferating cell nuclear antigen PCNA, thereby inhibitingprocessive DNA synthesis (18).

Because p21 was strongly induced in TPA-treated SkBr3cells, and because constitutive c-Myc overexpression in se-lected SkBr3 cells conferred resistance to TPA (16), we hy-pothesized that the inhibition of p21 expression may berequired for c-Myc deregulation of growth arrest and for itseffect to induce DNA synthesis in such quiescent cells.

We generated a c-Myc-expressing adenovirus (Ad-cMyc)to more easily study the effects of c-Myc protein in humancells. We show that an Ad-cMyc infection of either SkBr3 orLNCaP cells can overcome TPA-induced growth arrest. In-terestingly, c-Myc overexpression significantly inhibited p21expression in cells induced to enter into S phase. We ex-plored the significance of this inhibition by examining thekinetics of p21 expression in Ad-cMyc as compared withAd-E2F-1-infected cells. We further investigated the mech-anism of this inhibition and found evidence for transcriptionalrepression of p21 expression by c-Myc. Ad-p21/Ad-cMyccoinfection of SkBr3 cells revealed that p21-mediatedgrowth arrest was dominant over c-Myc’s effect to promoteDNA synthesis. The results suggest that the inhibition of p21expression may contribute to c-Myc-dependent S-phase en-try, possibly in cells in which growth arrest is p21-dependent.

Received 6/12/98; revised 2/9/99; accepted 3/3/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 K. O. M. is supported by the National Research Service Award Predoc-toral Fellowships for Minority Students. W. S. E-D. is an Assistant Inves-tigator of the Howard Hughes Medical Institute.2 To whom requests for reprints should be addressed, at Laboratory ofMolecular Oncology and Cell Cycle Regulation, Howard Hughes MedicalInstitute, University of Pennsylvania School of Medicine, Philadelphia, PA19104. Fax: (215) 573-9139; E-mail: weldeir@hhmi.upenn.edu.

3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-ace-tate; MOI, multiplicity of infection; BrdUrd, bromodeoxyuridine; DAPI,49,6-diamidino-2-phenylindole; Ad, adenovirus.

223Vol. 10, 223–230, April 1999 Cell Growth & Differentiation

ResultsCell Cycle Deregulation and Induction of Apoptosis afterc-Myc Overexpression in Human Cells. We generated ahuman c-Myc-expressing replication-deficient E1-deletedAd5 recombinant adenovirus (Ad-cMyc) to investigate theeffects of c-Myc on proliferation of human cells. The DNAsequence of the newly cloned human c-Myc cDNA wasverified. Fig. 1A shows that human c-Myc protein is easilydetected by Western analysis after infection of normal hu-man lung fibroblasts (WI38) as well as human breast (SkBr3),ovarian (SkOV3), lung (H460), and colon (SW480, HCT116)cancer cells. The mobility of the exogenous c-Myc coincidedwith that of the endogenous c-Myc protein (Fig. 1A, compareAd-LacZ versus Ad-cMyc-infected SkBr3 and H460 cells).Human c-Myc protein was also detected after the infection ofNIH3T3 mouse fibroblasts, used initially because of the lackof cross-reactivity between the antihuman c-Myc antibodyand mouse c-Myc protein. The level of c-Myc expressioncorrelated with both adenovirus infectivity (not shown) andMOI (Fig. 1B). We then confirmed that the overexpression ofc-Myc was primarily in the nucleus of Ad-cMyc-infected cells(Fig. 1C).

To determine whether the overexpressed c-Myc proteinafter Ad-cMyc infection has biological activity, we studied its

effects on the WI38 normal human lung fibroblast cell line.c-Myc overexpression has been previously shown to induceapoptosis of serum-deprived rat embryo fibroblasts (19).After 2 days of incubation in serum-deprived media afterAd-cMyc infection, the WI38 cells were stained with DAPI toexamine nuclear morphology and DNA integrity (Fig. 2). Ad-cMyc-infected and, subsequently, serum-deprived WI38 un-derwent massive nuclear fragmentation (Fig. 2, B and D) ascompared with Ad-LacZ-infected cells (Fig. 2, A and C).There are no DAPI(1) cells in Fig. 2C because those WI38cells were viable (same field in Fig. 2A), and the cell mem-branes were not permeabilized. Thus, the c-Myc-dependentapoptosis phenotype was recapitulated after the Ad-cMycinfection of human cells. To further show that this was aneffect of c-Myc overexpression in the WI38 cells, we per-formed immunohistochemical staining on cells infected witheither Ad-cMyc or Ad-LacZ under the same conditions (Fig.2, E and F). As expected, those cells infected with Ad-cMycshowed overexpression of the c-Myc protein as comparedwith those cells infected with Ad-LacZ. Ad-cMyc infection ofWI38 cells seemed to have a small effect on DNA synthesisinduction as compared with Ad-LacZ (Fig. 2G); the majoreffect after Ad-cMyc infection of WI38 cells was apoptosis(Fig. 2D). We further studied the effects of c-Myc overex-

Fig. 1. Overexpression of c-Myc in Ad-cMyc-infected mammalian cells. A, cell lines (as indicated) were infected using a MOI of 200 with either Ad-LacZ(“1” as indicated) or Ad-cMyc (“1” as indicated). Immunoblotting with anti-c-Myc antibody was performed as described in “Materials and Methods.” B,Western blot analysis of SkBr3 (upper panel) and NIH3T3 (lower panel) cell lines after infection with increasing MOIs (as indicated) of either Ad-cMyc orAd-LacZ (as indicated). C, Immunochemical analysis of c-Myc protein expression in SkBr3 cells after infection with either Ad-cMyc or Ad-LacZ (MOI 50).After a 1-h infection, these cells were treated with TPA for 30 h before immunocytochemistry.

224 c-Myc in TPA-sensitive Human Cancer Cells

pression on subsequent molecular events involved in cellcycle control.

c-Myc Bypass of TPA-induced Growth Arrest Is Asso-ciated with Decreased p21WAF1/CIP1 Expression. Becausewe previously observed that p21 levels are increased inTPA-induced growth arrest of SkBr3 cells (Fig. 4B; Ref. 16),we examined p21 protein expression levels after Ad-cMycinfection of these cells (Fig. 3A). We noted a significantinhibition in p21 expression in Ad-cMyc-infected as com-pared with either Ad-LacZ- or mock-infected TPA-arrestedSkBr3 cells. There was no increase in p27 levels in TPA-treated SkBr3 cells (Fig. 3B).

To determine whether or not this observation was limitedto only one cell line, the prostate cancer cell line LNCaP wasalso used. We first confirmed that this cell line was TPA-sensitive by examining p21 expression levels after exposureto TPA (Fig. 3C; Ref. 16). After showing that the p21 proteinexpression levels are increased after TPA treatment, we ex-amined the effects of Ad-cMyc infection (Fig. 3D). As seen inthe breast cancer cell line (Fig. 3A), we observed a significantinhibition of p21 protein expression in the c-Myc-overex-pressing TPA-treated prostate cancer cells (Fig. 3D).

We further investigated the kinetics of the TPA-inducedarrest in SkBr3 cells (Fig. 4). Addition of TPA to the SkBr3cells led to growth arrest detected as early as 12 h (Fig. 4A).The expected increase in p21 expression was observed by

4 h after the exposure of the cells to TPA, and this increasewas sustained for at least 24 h (Fig. 4B). Increased p21expression preceded inhibition of DNA synthesis after TPAtreatment of SkBr3 cells.

To determine whether c-Myc overexpression could inter-fere with TPA-induced growth arrest, Ad-cMyc infection wasused to constitutively overexpress c-Myc in SkBr3 cells (Fig.4C). Ad-cMyc infection of SkBr3 cells seemed to rescuethese cells from TPA-induced growth arrest (Fig. 4C; com-pare Ad-cMyc- versus Ad-LacZ-infected cells). These resultsdemonstrate in a human cancer cell line that the exogenousoverexpression of c-Myc could deregulate cell cycle control,as evidenced by the induction of DNA synthesis despite theTPA-mediated growth-arrest signal. These findings also sug-gest that the inhibition of p21 expression may occur duringabnormal c-Myc-induced S-phase entry.

In order to investigate the possibility that the effect on p21may be simply due to the progression of cells into S phase,we compared the effects of overexpression of E2F-1 andc-Myc (Fig. 4C). E2F-1 overexpression also rescued theSkBr3 cells from the TPA-induced growth arrest. However,the expression of p21 was not down-regulated until theAd-E2F-1-infected cells entered S phase (compare Fig. 4, Cand D).

To further investigate the effect of E2F-1 overexpressionon inducing S-phase entry, we analyzed BrdUrd incorpora-

Fig. 2. Induction of apoptosis and DNA synthesis deregulation after Ad-cMyc infection of WI38 cells. WI38 cells were infected by either Ad-LacZ (A, C,E) or Ad-cMyc (B, D, F) and then incubated at 37°C in DMEM without serum for 2 days. The cells were then analyzed by phase- (A, B) or UV-microscopy(C, D) after DAPI staining. The same high-power fields are shown in panels A and C or B and D. E, F, Immunochemical analysis of c-Myc protein expression.G, WI38 cells initially serum-starved for 48 h, subsequently infected with a MOI of 250 using either Ad-cMyc (Lane 3) or Ad-LacZ (Lane 2), were analyzed30 h later after an additional 6-h incubation in the presence of [3H]thymidine. Lane 1 is mock-infected cells that were kept in serum-deprived conditions,and in Lane 4, cells were restimulated with 10% serum.

225Cell Growth & Differentiation

tion in parallel with p21 immunohistochemistry (Fig. 5). Weobserved a significant increase in BrdUrd-incorporating cellsover time, whereas there was little decrease in p21 staininguntil 20 h postinfection. At 16 h, the majority of Ad-E2F-1-infected SkBr3 breast cancer cells incorporated BrdUrd (Fig.5N) while continuing to overexpress p21 protein (Fig. 5M). By20 h, only a small minority of Ad-E2F-1 cells expressed p21(Fig. 5S), whereas the vast majority of the cells continued toincorporate BrdUrd (Fig. 5T). These results are consistentwith the interpretation that E2F-1 deregulation of S-phaseentry probably acts downstream of p21 and that the inhibi-tion of p21 expression in response to E2F-1 may be a con-sequence of cell cycle progression. These data are in con-trast to the c-Myc situation in which the suppression of p21expression seems to precede entry into S phase in Ad-cMyc-infected TPA-treated cells (Fig. 4, C and D). In the case ofc-Myc, a clear decrease in p21 staining was observed by16 h postinfection (Fig. 5O), whereas there was no sign ofBrdUrd incorporation by this time point (Fig. 5P). Theseresults indicate that the down-regulation of p21 expressionby overexpression of c-Myc is probably not a consequenceof S-phase progression but may be part of the primary mech-anism by which c-Myc induces S-phase entry. We furtherexamined the mechanism of c-Myc-dependent inhibition ofp21 expression and the requirement of this inhibition for cellcycle deregulation.

Transcriptional Repression of p21WAF1/CIP1 Expressionby c-Myc. There is evidence for transcriptional, posttran-scriptional, and posttranslational control of p21 expression incellular growth control pathways (20–22). Using Northernanalysis, we compared p21 mRNA levels in Ad-cMyc- andAd-LacZ-infected TPA-treated SkBr3 cells (Fig. 6A). Wefound that p21 mRNA levels were significantly lower in Ad-cMyc- as compared with Ad-LacZ-infected cells.

In order to determine whether c-Myc might transcription-ally repress p21 expression through an effect on the p21promoter, we examined the effects of human c-Myc expres-sion on expression of a luciferase-reporter gene linked to thehuman p21 promoter (Fig. 6B). We found that the transfec-tion of c-Myc repressed the transcription from the p21 pro-moter by two- to three-fold as compared with a controltransfection driving antisense c-Myc expression. These ex-periments were carried out in SW480 human colon carci-noma cells because of their relatively high transfection effi-ciency (30–50%). To confirm that p53 DNA-binding sites arenot required for repression by c-Myc, a p21-promoter frag-ment lacking the potential for regulation by p53 was cotrans-fected with c-Myc into (mutant p53-expressing) SW480 cells(Fig. 6B, 4-Luc). The results further suggest that the inhibitionof p21 expression after overexpression of c-Myc may occurin part through the repression of transcription.

Inhibition of p21WAF1/CIP1 Expression Is Required forInduction of DNA Synthesis by c-Myc. We further exam-ined the possibility that the inhibition of p21 expression maybe required for S-phase deregulation by oncogenic c-Mycprotein. We hypothesized that overriding the inhibition of p21expression by c-Myc (by constitutively overexpressing p21)should prevent entry into S phase if this inhibition is criticalfor c-Myc function. Coinfection of SkBr3 cells (at identicalMOIs) by Ad-cMyc and Ad-p21 resulted in the suppressionof c-Myc-dependent S-phase entry (Fig. 7).

These results support the idea that deregulation of S-phase entry by c-Myc may be suppressed by exogenous p21overexpression, and that the observed c-Myc-dependentinhibition of endogenous p21 expression may be required forc-Myc deregulation of DNA synthesis control, especially insituations in which p21 mediates growth arrest.

DiscussionWe have identified a novel downstream effect of oncogenicc-Myc to inhibit expression of the cell cycle inhibitorp21WAF1/CIP1. The deregulation of TPA-induced growth ar-rest by c-Myc suggests that c-Myc overexpression may besufficient to induce S-phase entry in a growth-arrested hu-man cancer cell line. We provide evidence for the transcrip-tional repression of p21 expression by c-Myc precedingentry into S phase. Finally, we find that constitutive p21overexpression inhibits deregulation of DNA synthesis byc-Myc, which suggests that the inhibition of p21 expressionby c-Myc may contribute to its cell cycle promoting effect.

In addition to cell cycle deregulation, the inhibition of p21expression could lead to the loss of other p21-dependentfunctions in c-Myc-overexpressing cells. p21 has been foundto be a potent suppressor of cellular transformation (23), andthe loss of p21 expression has been suggested to contribute

Fig. 3. The inhibition of p21 expression in cell cycle-deregulated c-Myc-overexpressing cells. A, Western blot analysis of p21 expression in SkBr3cells mock-infected (Lanes 1 and 2) or infected with a MOI of 25 of eitherAd-LacZ (Lane 3) or Ad-cMyc (Lane 4) and incubated for 30 h in theabsence (2) or presence (1) of TPA as indicated. B, Western blot analysisof p27 expression in SkBr3 cells for 30 h in the absence (2) or presence(1) of TPA. C, Western blot analysis of p21 expression in LNCaP cells for30 h in the absence (2) or presence (1) of TPA. D, Western blot analysisof c-Myc and p21 expression in LNCaP cells infected with either Ad-LacZ(Lane 1) or Ad-cMyc (Lane 2) at MOIs of 2 for 30 h in the presence of TPA.Actin expression is used as a loading control for each blot.

226 c-Myc in TPA-sensitive Human Cancer Cells

to ras- (24) and E1A-induced (25) transformation. It is pos-sible that in vivo, c-Myc may provide a signal to suppressp21 as a transformation-predisposing event that could thenallow other oncogenes such as ras to complete the transfor-mation. In some studies, p21 has been found to protectmammalian cells against apoptosis (26–28). Thus, the loss ofp21 expression could also provide a permissive cellular en-vironment for c-Myc-induced apoptosis. p21 has also beenimplicated in the control of a fundamental aspect of cell cyclecontrol, the coupling of S and M phases to ensure that cellsdo not reduplicate their DNA if they have not undergonemitotic division (28). Thus, the c-Myc suppression of p21expression could predispose c-Myc-overexpressing cells toabnormal S-M coordination.

Our results are somewhat different from recent observa-tions in rat embryo fibroblast cells, suggesting that c-Mycalone was ineffective in inducing S phase (29) and that raswas required (but also ineffective when overexpressed alonein rat embryo fibroblast cells). One possible explanation forthese differences is that our studies were carried out inhuman cancer cells. In this regard, it was previously shown(30) that SkBr3 cells express increased levels of the ras-related rho proteins, in particular rhoB, and are highly sen-sitive to growth inhibition by inhibitors of tyrosine kinaseactivity (31). Thus, it is possible that in our experiments,c-Myc was sufficient to induce S-phase entry because theras pathway may be deregulated in SkBr3 cells. Perhaps not

surprisingly then, in the serum-starvation-arrested WI38 nor-mal lung fibroblasts, there was a poor induction of DNAsynthesis after Ad-cMyc infection.

A possible explanation of our results is that the inhibition ofp21 expression after c-Myc overexpression may be an indi-rect effect of cell cycle progression. We addressed this issueby inducing S-phase entry by E2F-1-overexpression. Theresults in Figs. 4 and 5 suggest that there is a fundamentaldifference in the mechanism of cell cycle deregulation byc-Myc versus E2F-1. In TPA-arrested breast cancer cells,Ad-E2F-1 infection led to early S-phase entry (seen by 12 hin some cells; Fig. 5J), whereas the majority of the cellsoverexpressed p21. By 16 h, the vast majority of Ad-E2F-1infected cells were in S phase as assessed by BrdUrd incor-poration (Fig. 5N) and continued to overexpress p21. Only by20 h, were p21 levels decreased in Ad-E2F-1 infected TPA-treated cells. These results suggest that in the case of E2F-1,the suppression of p21 expression is not required for dereg-ulation of S-phase entry. In the case of c-Myc, the resultsdemonstrate that the suppression of p21 expression occursbefore S-phase entry. Thus, in TPA-sensitive cells, TPA in-duces a cell cycle arrest in both G1 and G2

4 associated withthe induction of p21 (but not p27) protein expression (Figs. 3and 4) and the inhibition of c-Myc expression (16). In such

4 K. O. Mitchell and W. S. El-Deiry, unpublished data.

Fig. 4. TPA-induced growth ar-rest and rescue by c-Myc over-expression. In A, SkBr3 cellswere incubated in the presence(1) or absence (2) of TPA andanalyzed at the indicated num-ber of hours after treatment, in-cluding 4 h of incubation in thepresence of [3H]thymidine. B,Western blot analysis of p21 ex-pression in SkBr3 cells incu-bated in the presence (1) or ab-sence (2) of TPA for the numberof hours indicated. In C, after16 h of TPA exposure, SkBr3cells were infected with Ad-LacZ, Ad-cMyc, or Ad-E2F-1(MOI 25), and the extent of newDNA synthesis was analyzed by[3H]thymidine incorporation atthe indicated number of hourspostinfection (HPI). These timesalso included the 4-h incubationwith [3H]thymidine. D, Westernblot analysis of c-Myc, E2F-1,and p21 expression in TPA-treated SkBr3 cells infected witheither Ad-LacZ, Ad-cMyc, or Ad-E2F-1 and harvested for analysisat the indicated times postinfec-tion. Lanes 1–3: 8 h, Lanes 4–6:16 h, Lanes 7–9: 24 h, and Lanes10–12: 32 h.

227Cell Growth & Differentiation

situations in which growth arrest is associated with elevatedp21 expression, the introduction of constitutive c-Myc over-expression: (a) deregulates the arrest; and (b) suppressesp21 expression before S-phase entry. Our results suggestthat the suppression of p21 expression is an early event thatfollows c-Myc overexpression and argue that it may be a

necessary step in cell cycle deregulation, at least in TPA-arrested p21-overexpressing SkBr3 breast cancer cells.

Materials and MethodsCell Lines and Culture Conditions. Early passage WI38 normal humanlung fibroblasts, SkOV3 human ovarian carcinoma, SkBr3 human breast

Fig. 5. The kinetics of E2F-1 rescue of TPA-induced growth arrest in SkBr3 breast cancer cells. A, C, E, G, I, K, M, O, Q, S, Immunochemical analysis ofp21 protein expression after mock infection (A) or infection with Ad-LacZ (C, G, K, Q; MOI 25), Ad-E2f-1 (E, I, M, S; MOI 25), or Ad-cMyc (O; MOI 20) asindicated. SkBr3 cells were incubated in the presence of TPA for 16 h before a 1-h infection and then continued treatment with TPA for the indicated numberof hrs before immunocytochemistry. B, D, F, H, J, L, N, P, R, T, BrdUrd incorporation in SkBr3 cells after the mock infection (B) or the infection with Ad-LacZ(D, H, L, R; MOI 25), Ad-E2F-1 (F, J, N, T; MOI 25), or Ad-cMyc (P; MOI 20) as indicated. SkBr3 cells were treated as described above and BrdUrdincorporation was examined under fluorescence microscopy at the indicated number of hours postinfection. The times also include 4 h of incubation withBrdUrd.

228 c-Myc in TPA-sensitive Human Cancer Cells

cancer cells, and LNCaP human prostate cancer cells were obtained fromAmerican Type Culture Collection and cultured under the recommendedconditions. The human non-small cell lung cancer cell line H460 wasprovided by S. B. Baylin (Johns Hopkins University, Baltimore, MD) andthe human colon cancer cell line HCT116 was provided by B. Vogelstein(Johns Hopkins University). SW480 human colon carcinoma cells wereobtained from the Cell Center at the University of Pennsylvania (Philadel-phia, PA). For serum deprivation experiments, WI38 cells were incubatedin media containing 0.5% fetal bovine serum for 48 h before adenovirusinfection as described below. Treatment with the phorbol ester TPA wascarried out using 50 ng/ml for different lengths of time as indicated in thefigure legends.

Adenovirus Preparation and Infection. A human c-Myc-expressingAd5 adenovirus recombinant was generated as previously described for

p53 and p21 (32, 33). The c-Myc cDNA was amplified from H460 total RNAby reverse transcription-PCR using the following primers and PCR con-ditions: forward primer 59-ATACGCGGATCCACCATGCCCCTCAACGTT-AGCTTCAC-39; and reverse primer 59-GCGTATCCTAGGTTACGCACAA-GAGTTCCGTAGCT-39; amplification for 35 cycles of denaturation at 94°Cfor 30 s, annealing at 60°C for 1 min, and extension at 72°C for 2 min. ThecDNA was subcloned into the pCRII vector (Invitrogen) and completelysequenced. The c-Myc cDNA was in vitro translated, and the resultingprotein (;Mr 64,000) was easily recognized by anti-c-Myc antibody byWestern analysis (not shown). After the generation of Ad-cMyc, expres-sion of c-Myc protein was documented by Western analysis and immu-nocytochemistry (Fig. 1) as described below. Ad-E2F-1 was provided byDr. Joseph R. Nevins (Duke University, Durham, NC). Adenovirus titersand infections were carried out as described previously (33).

Western Analysis and Immunocytochemistry. Protein lysates wereprepared, and Western analysis was performed as previously described(32), with the following modifications. The Lumigen PS-3 detection rea-gent (ECL1Plus, Amersham) was used according to the manufacturer’srecommendations. Antihuman c-Myc monoclonal antibody 9E10 clonewas obtained from Santa Cruz Biotechnology and the antihuman WAF1monoclonal antibody Ab1 was obtained from Calbiochem. Immunocyto-chemistry after adenovirus infection was performed as previously de-scribed (34).

DNA Synthesis and Apoptosis Assays. The extent of new DNA syn-thesis was assessed by [3H]thymidine incorporation assays (25) andBrdUrd incorporation (35) as described. DAPI staining of nuclear mor-phology to evaluate Ad-LacZ- or Ad-cMyc-infected cells for chromosomalDNA fragmentation was carried out as described previously (34).

Transfection and Luciferase Assays. SW480 cells were transfectedusing human p21-promoter luciferase-reporters and either pCMV-cMyc-Sor pCMV-cMyc-AS plasmids, as indicated in the legend to Fig. 4. ThecMyc plasmids drive expression from the human c-Myc cDNA, either inthe sense (pCMV-cMyc-S) or antisense (pCMV-cMyc-AS) orientation,driven by the immediate early promoter of CMV. Expression of humanc-Myc protein was demonstrated (not shown) after the transfection ofNIH3T3 with the sense construct, followed by Western analysis usingantihuman c-Myc antibody as described above. Transfections and luci-ferase assays were performed as described previously (36). NIH3T3 cellswere transfected using the Lipofectamine reagent (Gibco-Bethesda Re-search Laboratory) as recommended by the manufacturer.

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Fig. 6. Transcriptional repression of p21 in c-Myc-overexpressing cells. A, Northern analysis of p21 mRNA levels in SkBr3 cells following infection at a MOIof 25 with either Ad-LacZ or Ad-cMyc (as indicated) and incubation in the presence of TPA for 30 h. B, SW480 cells were cotransfected at a 3;1 ratio ofeither pCMV-c-Myc-Sense (hatched bars) or Antisense (solid bars) expression plasmid and either 0-Luc or 4-Luc (as indicated, see schematic). After 16 hof incubation, the transfected cells were harvested and assayed for luciferase activity. S1 and S2, the two p53 DNA-binding sites located in thep21-promoter luciferase-reporter 0-Luc.

Fig. 7. Constitutive overexpression of p21 leads to growth arrest and isdominant over c-Myc deregulation of DNA synthesis. Analysis of DNAsynthesis in TPA-treated SkBr3 cells that were coinfected with differentcombinations of Ad-cMyc, Ad-LacZ, and Ad-p21. Each virus was used ata MOI of 15, whether only one virus or two viruses were used for infection.After 33 h of TPA treatment the cells were harvested and analyzed for[3H]thymidine incorporation as described in “Materials and Methods.”

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