(CANCER RESEARCH 47. 6.110-6314, December 1, 1987]

Regulation of N-myc Transcript Stability in Human Neuroblastoma andRetinoblastoma Cells1

Christopher M. Amy2 and James C. Bartholomew3

laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

ABSTRACT

Several studies have shown that neuroblastoma and retinoblastomatumor cells often have elevated N-myc mRNA levels compared to normaladult neuronal or retinal tissues, and it has been suggested that increasedexpression of this protooncogene may play an important role in tumori-genesis or malignant progression in cells of neural origin. We have studiedthe effect of protein synthesis inhibitors on the N-myc mRNA levels inY79 retinoblastoma and LA-N-5 neuroblastoma cells. We showed thatwhen new RNA synthesis was inhibited by actinomycin D the levels ofexisting N-m>'CmRNA fell rapidly in both cell lines relative to totalcytoplasmic RNA. The half-life for N-myc mRNA was approximately 30min. Inhibition of protein synthesis by interfering with polypeptide elongation or by inhibiting initiation of protein synthesis increased the levelsof N-m>'cmRNA at least 3-fold after 4 hours. Nuclear runoff transcriptionexperiments showed that the protein synthesis inhibitors did not alter N-myc transcription rates. Combined actinomycin D treatment and treatment with protein synthesis inhibitors indicated that this increase in N-myc transcript levels was due to an increase in the N-myc mRNAlifetimes. Thus, N-myc transcript levels increased because they weremore stable in protein synthesis-inhibited cells. Protein synthesis inhibition also increased c-m_rcmRNA levels in 111 (ill human promyelocyticleukemia cells, but no increase was seen in the relatively low level of c-myc mRNA in protein synthesis-inhibited neuronal tumor cells. Theseresults support the hypothesis that the regulation of \-myc in theseneuronal and retinal tumor cells is similar to that of c-myc in other tumortypes.

INTRODUCTION

N-myc is a member of the myc gene family related in sequenceand genomic organization to the v-myc oncogene and the c-myc and L-myc protooncogenes ( 1). N-myc, L-myc, and c-myccan cooperate with the raígene to transform rat embryo fibro-blast cells in culture (1-3), suggesting a similar role for thesegenes in tumorigenesis. Increased c-myc expression is correlatedwith a wide variety of cell and tumor types (4). Both L-myc andN-myc, however, appear to be expressed in a more limited rangeof cell types (1,5, 6). Increased N-myc expression has beenshown to occur in neuroblastoma (7), retinoblastoma (8), smallcell lung carcinoma (9), and Wilms' tumors (10). Except inWilms1 tumors (I), the N-myc gene is often amplified in these

tumors, and Seeger et al. (Il ) reported that amplification of theN-myc gene is associated with rapid progression of neuroblas-

tomas and concluded that multiple copies of this gene may playa role in determining tumor aggressiveness.

Understanding the mechanisms used by cells to regulatesteady-state levels of myc mRNA is central to determiningwhether (and how) the myc gene products influence malignanttransformation of cells. The regulation of c-myc expression hasbeen shown to involve both transcriptional and posttranscrip-

Received 5/21/87; revised 8/31/87; accepted 9/10/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the Office of Energy Research, Office of Healthand Environmental Research. Health Effects Research Division of the UnitedStates Department of Energy under Contract DE-AC03-76SF00098.

2Present address: Children's Hospital Oakland Research Institute, Oakland,

CA 94609.1To whom requests for reprints should be addressed.

tional steps (12). In the latter case, protein synthesis inhibitionwas shown to increase the lifetime of c-myc mRNA (12).Decreased expression of N-myc precedes the differentiation ofhuman neuroblastoma cells induced by retinoic acid (13) in amanner similar to the down-regulation of c-myc during chemically induced differentiation of HL-60 promyelocytic leukemiacells (14, 15). Interestingly, there were no differences in the lowlevels of c-myc mRNA in retinoic acid-treated and controlneuroblastoma cells, suggesting that in these cells N-mycexpression was regulated by a different mechanism than c-myc(13). As a further comparison of c-myc and N-myc, we haveinvestigated how N-myc transcript levels are affected by proteinsynthesis inhibition in cultured human cells, particularly theY79 cell line isolated from a retinoblastoma tumor and the LA-N-5 cell line from a neuroblastoma tumor. These cell lines have15-50 copies of this gene encoding 50-200 times as much N-myc mRNA as the neuroblastoma line SK-N-SH which hasonly one genomic copy of the N-myc (16). We report here thatthe half-life of N-myc mRNA in Y79 and LA-N-5 cells is shortand that the lifetimes of this mRNA can be lengthened byinhibiting protein synthesis. This increase in N-myc messageoccurs whether inhibitors of protein synthesis initiation (pac-tamycin) or inhibitors of different stages of polypeptide elongation (cycloheximide, emetine, or anisomycin) were added tocells. Our data suggest that a posttranscriptional mechanismexists for regulating the lifetime of N-myc transcripts in Y79and LA-N-5 cells in much the same manner as has beendescribed for c-myc mRNA levels in other cell types (12).

MATERIALS AND METHODS

Cell Lines and Plasmids. Y79 retinoblastoma and LA-N-5 and SK-N-SH neuroblastoma cell lines were obtained from Wen-Hwa Lee, andHL-60 promyelocytic leukemia cells were from the American TypeCulture Collection. Each cell line was grown in RPMI (G1BCO) supplemented with 10% fetal bovine serum and was transferred to freshgrowth medium 24 h before the addition of inhibitors to cell cultures.

The pNB-1 N-myc human genomic probe was from M. Bishop; thepWLSOl plasmici constructed by Wen-Hwa Lee contains a 3.8-kilobasehuman genomic fragment homologous to the pNb-1 probe DNA. Theph-mj'c (human c-myc) was obtained from A. Levinson; pT24-C3(human c-Ha-ros), pNRSac (human N-raj), and pc-fos (human c-fos)were from the American Type Culture Collection.

Inhibitor Treatment. Actinomycin D, a-amanitine, cycloheximide,anisomycin, and emetine were obtained from Sigma Chemical Company; pactamycin was from Upjohn. Each of the inhibitors was dissolved in sterile water and added to 10 ml of growth medium in avolume of SOnI or less to a final concentration which inhibited proteinsynthesis by greater than 90%.

Rate of Transcription in Isolated Nuclei. Nuclear runoff transcriptionassays were used to measure the rate of transcription of specific genesusing the procedure described by Greenberg and Ziff (17). At the endof the runoff reaction, nuclear RNA was isolated by treatment withDNase and proteinase K as described for cytoplasmic RNA isolation(see below). The amount of each labeled RNA homologous to probesfor different genes was determined by hybridization to 5-jig aliquots ofplasmid DNA linearized with EcoRI. DNA probes slot-blotted ontonitrocellulose filters were hybridized to the same number of cpm of

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REGULATION OF N-myc mRNA STABILITY

[32P]RNA from each runoff assay for 48 h at 42°Cin 50% formamide,5 x SSC," 20 raw phosphate buffer (pH 6.8), 1% SDS, and 250 Mg/mlyeast tRNA at 42"C for 24 h. After hybridization, each filter waswashed twice for I h with 2 x SSC at 65 "C and exposed to Kodak X-

Omat AR film for autoradiography. Individual sections of filters werecounted in a liquid scintillation counter to measure the amount oflabeled RNA hybridizing with each plasmid DNA.

Cytoplasmic RNA Isolation. Total cytoplasmic RNA was isolatedfrom tissue culture cells using a modification of the procedure ofFavaloro et al. (18). Cells were harvested from monolayer cultures byscraping the cells off the dishes or from suspension cultures by ceritrifugation at 800 x g. Cell pellets were washed in cold phosphate-buffered saline and resuspended in 200 Ml/100-mm dish [approximately107cells] of ice-cold lysis buffer [10 mM Tris-HCI, pH 8.3-2 mM vanadyl

ribonucleoside complex (Bethesda Research Laboratory)]. Twenty n\ of10% Nonidet P-40 detergent were added dropwise with vortexing. Thesamples were placed on ice for 5 min, and nuclei from the lysed cellswere pelleted by centrifugation at 15,000 x g for 5 min at 4'C. The

supernatant was removed to a fresh tube and mixed with an equalvolume of 2 x proteinase K buffer [200 mM Tris-HCI (pH 7.5), 25 mMEOT A, 0.3 M NaCl, and 2% SDS] to which were added 200 Mg/ml ofproteinase K. After 30 min at 37°C,these samples were extracted with

phenol/choloroform, and the RNA was concentrated by precipitationwith 2.5 volumes of ethanol at -20°C. Each RNA pellet was resus

pended in water and quantified by its absorbance at 260 nm; approximately 80-100 jig of RNA were obtained from IO7Y79 cells.

Electrophoresis and Quantification of Specific RNA Transcripts. Ali-quots (10 ¿/g)of cytoplasmic RNA were electrophoresed through form-aldehyde/agarose denaturing gels as described by Maniatis et al. (19)and transferred to nitrocellulose by the method of Thomas (20). Replicate 10-Mgaliquots of the same RNA were denatured and dotted ontonitrocellulose using a 96-well vacuum manifold. RNA was immobilizedon the nitrocellulose filters by baking, prehybridized using the solutiondescribed above with 250 ¿ig/mlof salmon sperm DNA instead of yeasttRNA, and hybridized with 10 ng/ml of plasmid DNA labeled with 32Pby nick translation to a specific activity of 1-2 x 10" cpm/pg of DNA.After 48 h at 42°C,the filters were washed twice for 15 min in 2 x

SSC-0.1% SDS at room temperature, twice in 0.1 x SSC-0.1% SDSfor 15 min at room temperature, and then once for 20 min at 50°Cin

0.1 x SSC-0.1% SDS. After autoradiography, the blotted RNA wasquantified by counting each dot in a liquid scintillation counter. All ofthe hybridizable material was eliminated by treatment with RNaseindicating that these samples were free of DNA which hybridized withthese probes. For quantification of RNA in blotted gels, autoradiogramsof filters hybridized with specific probe DNA was scanned with a GS300 scanning densitometer (Hoefer Scientific Instruments). Sizes ofmRNA transcripts were estimated by comparison with the 18S (1.8-kilobase) and 28S (5-kilobase) rRNA bands stained with ethidiumbromide prior to gel blotting or by comparison to X Ili/unii fragmentswhich were end-labeled, denatured, and electrophoresed along with theRNA as described by Maniatis et al. (19).

RESULTS

Increase in N-myc RNA Levels in Cells Treated with ProteinSynthesis Inhibitors. Fig. 1 shows the time course of the effectof three protein synthesis inhibitors on the level of N-myc RNAin the pool of total RNA from Y79 retinoblastoma cells. Allthree inhibitors produced similar increases of about 3-fold inthe N-myc RNA after 4 h, and the untreated cells showed nochanges in N-myc RNA over the same period. Cycloheximidehas no measurable effect on the comparatively low N-myc RNAlevels in SK-N-SH cells after 2 h of treatment (Fig. 1).

We next investigated whether the expression of other onco-genes in neuronal and nonneuronal tumor cells was increasedin response to these protein synthesis inhibitors. Table 1 pre-

300

250

200-

150

100

50

hrs after inhibitor addition

Fig. 1. Levels of N-myc RNA in Y79 cells treated with protein synthesisinhibitors. Anisomycin (70 niu; V), eycloheximide (70 UM;D). or emetine (50 ¡IM;A) were added to Y79 cells, and dishes of treated and untreated cells (x) wereharvested for cytoplamsic RNA isolation after 1, 2, 3, and 4 h. RNA was alsoisolated from an untreated culture of SK-N-SH cells (O) and from SK-N-SH cellstreated for 2 h with eycloheximide (70 JIM; •).Each RNA was dotted ontonitrocellulose and hybridized with "P-labeled pNB-l N-myc DNA (1.05 x 10'cpm/ml) as described in "Materials and Methods." Individual dots were counted,and the results are presented as the percentage of (32P]DNA bound to each slotof 10 ng of RNA from inhibitor-treated cells compared to untreated cells. Theseresults are representative of 4 separate experiments.

Table 1 Dolblotted RNA probed with oncogenaTotal cytoplasmic RNA from Y79, LA-N-5, and HL-60 cells were isolated

after a 1-h exposure to actinomycin D or a 3-h exposure to 2 /IM pactamycin or70 //M anisomycin. The RNA was purified and dotted onto nitrocellulose in 10-fig aliquots and hybridized with either pWLSOl (1.03 x 10' cpm/Vg). pT24-C3(6.1 x 10' cpm/>ig), or ph-myc (1.22 x 10* cpm/Vg) DNA as described in"Materials and Methods."

cpm/IO UKof totalcytoplasmicRNACellsY79LA-N-5HL-60TreatmentControlActinomycin

DPactamycinAnisomycinControlActinomycin

DPactamycinAnisomycinControlActinomycin

DPactamycinAnisomycinN-myc892014919021279363459._———c-Ha-ras464546523233374133375674c-myca——_———24717910831557

4The abbreviations used are: SSC, standard saline-citrate; SDS, sodium do-

decyl sulfate.

" Dots with hybridization equal to background levels.

sents data from RNA dots from several cell lines treated withinhibitors and probed for N-myc, c-Ha-ras, or c-myc sequences.For N-myc, the neuronally derived Y79 and LA-N-5 cells againshowed increases with protein synthesis inhibition. HL-60 pro-

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REGULATION OF N-myc mRNA STABILITY

myelocytic leukemia cells did not show a significant amount ofhybridization to the N-myc probe. HL-60 cells did, however,show a 4- to 6-fold increase in transcript levels in the presenceof protein synthesis inhibitors when RNA dots were probed forhuman c-myc sequences. RNA from neither of the neuronalcell lines showed detectable c-myc hybridization over background levels in this experiment. When the c-Ha-ras gene wasused to probe this RNA, essentially the same low levels ofcounts were obtained for each of the cell lines and treatmentconditions. Also, the RNA synthesis inhibitor actinomycin Dreduced the level of both N-myc and c-myc RNA in all casestested but had little effect on the level of c-Ha-ros (Table 1).

Cycloheximide, emetine, and anisomycin each inhibit theelongation of nascent protein chains and cause ribosomes to be"frozen" to the mRNA (21, 22). To examine whether the effect

of the protein synthesis inhibitors on N-myc mRNA levelswhich we observed was due to a physical shielding of themessage from degradation, we tested the effect of pactamycinon the levels of N-myc RNA in cells. Pactamycin, at theconcentrations used in this study (2 ^M). causes the inhibitionof protein synthesis by inhibiting initiation, followed by desta-bilization and dissociation of the polysomes (23). When pactamycin was added to Y79 and LA-N-5 cells, N-myc mRNAlevels rose by over 50% in each case (Table 1). Thus, shieldingof N-myc by ribosomes appears not to be responsible for theprotective effect since pactamycin has the same qualitativeeffect as the other protein synthesis inhibitors.

Lack of Effect of Inhibition of Protein Synthesis on Rate ofTranscription of N-myc. The effect of protein synthesis inhibitors shown in Fig. 1 could be the result of an action on the rateof transcription of the N-myc genes, an effect on the lifetime ofthe message, or both. We tested whether the protein synthesisinhibitors were affecting the transcription of the N-myc genesby using runoff transcription assays to measure transcriptionrates and compare them with the accumulation of mRNA inthe cytoplasm of the same cells. Nuclei for runoff transcriptionassays were isolated from Y79 and LA-N-5 neuroblastoma cellstreated with protein synthesis inhibitors for 30 min or 3 h. TheRNAs isolated from the cytoplasm of the same cells werepurified and separated on a denaturing gel, blotted, and probedfor N-myc RNA.

As the results in Fig. 2 show, the N-myc transcripts from thecytoplasms of Y79 and LA-N-5 cell lines had a size of about3.2 kilobases as reported in previous studies (16). There was,as expected, no detectable hybridization to transcripts in thecytoplasm of HL-60 promyelocytic leukemia cells probed withN-myc since HL-60 cells do not express N-myc (7) (Table 1).Quantification of the autoradiograms of the N-myc probe hybridized to RNA separated on gels and blotted onto nitrocellulose in Fig. 2 showed that the level of N-myc in Y79 cells wasincreased about 5-fold after exposure to anisomycin for 30 minor 3 h. LA-N-5 cells treated with cycloheximide showed a 2-fold increase over untreated LA-N-5 cells.

Although the levels of cytoplasmic N-myc RNA increased incells treated with protein synthesis inhibitors (Fig. 2), the nucleiisolated from these cells showed no changes in the rate of N-

myc transcription (Fig. 3). No new initiation of transcriptionoccurred in nuclei isolated under these conditions, and theamount of radioactivity incorporated into a specific RNA sequence in vitro is proportional to the number of nascent transcripts present at the time the nuclei were isolated (24). Theamount of labeled RNA hybridizing to each of the blotted DNAwas essentially the same after 20 and 40 min of incubation withlabeled DTP showing that runoff labeling of the RNA was

Northern Blot of N-myc RNA

Y79 HL60 LA-N-5

ino

JTO

OOü

TO

ü

—28 S

—18 S

1.0 4.5 5.3 0.0 6.9 14.3

Relative Intensity of N-myc BandFig. 2. N-m>'Ctranscripts in the cytoplasm of cells assayed for nuclear runoff

transcription. Cytoplasmic RNA from Y79 cells treated with anisomycin (ANI)and from LA-N-5 cells treated with cycloheximide (CH) were compared to RNAfrom untreated (CON) Y79. HL-60. and LA-N-5 cells. Ten /ig of each cytoplasmicRNA were electrophoresed on an agarose/formaldehyde gel, blotted, and probedfor N-myc mRNA. The migrations of the 18S ( 1.8 kilobases and 28S (5 kilobases)rRNA through the gel were determined by staining the gel with ethidium bromidebefore blotting.

complete after 20 min and that degradation of the RNA in thenucleus was insignificant (Fig. 3). Also, a-amanitine, an inhibitor of RNA polymerase II, inhibited virtually all of the hybridization to each of the plasmid DNAs probed with the RNAproduced during the runoff experiment from control nuclei.Other runoff experiments demonstrated that «-amanitine wassimilarly effective in eliminating almost all of the hybridizationto these probe DNAs in runoff transcription assays of cellstreated with protein synthesis inhibitors (data not shown).

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REGULATION OF N-myc mRNA STABILITY

CONTROL

Fig. 3. Nuclear runoff transcription assays.Labeled runoff transcripts from nuclei isolatedfrom untreated Y79 and LA-N-5 cells andfrom cells treated with protein synthesis inhibitors (70 *iM anisomycin or cycloheximide)were each hybridized to S linearized plasmidDNA dotted onto nitrocellulose as describedin "Materials and Methods." Runoff reactions

were performed for 20 or 40 min and an equalamount (4.2 x 10* cpm/ml) of labeled RNA

produced in each reaction was hybridized toeach set of DNA slots containing the followingplasmid DNA: N-myc (pWLSOl); c-myc (ph-myc); c-fos (pc-fos); c-Ha-ras (pT24-C3); andN-r«w(pNRSac).

(0<

0>JDO

ANISOMYCIN

0.5 hr 3 hrs

CON CH

3 hrs

pWL501

ph-myc

pc-fos

u- pT24-C3

pNRSac

20' 40' amanitin 20' 40' 20' 40' 20' 20'

Y79 LA-N-5

No differences in hybridization of runoff RNA from Y79cells with or without anisomycin treatment for 30 min or 3 hwere observed. Hybridization to the pWLSOl (N-myc) probe ineach of the treatment conditions showed the same number ofspecific mRNA in each sample. Likewise, labeled RNA isolatedfrom LA-N-5 cell nuclei treated for 3 h with cycloheximideshowed the same level of hybridization as RNA from untreatedcells. Thus, N-myc mRNA from untreated Y79 and LA-N-5cells appears to be transcribed at the same rate as from cellstreated with protein synthesis inhibitors. Other DNA-contain-ing sequences specific for the human c-myc, c-fos, c-Ha-ras, andN-ras also showed no significant changes in their rates of

transcription in cells treated with protein synthesis inhibitors.N-myc RNA Degradation in Cells Treated with Protein and

RNA Synthesis Inhibitors. The results shown in Fig. 3 suggestedthat the altered level of cytoplasmic N-myc RNA was not dueto the effect of protein synthesis inhibitors on the transcriptionof the N-myc gene. We therefore tested whether protein synthesis inhibitors were acting through an effect on the stabilityof the mRNA. RNA synthesis and/or protein synthesis inhibitors were added to Y79 cells for up to 90 min to determine howN-myc transcript levels increased with exposure to proteinsynthesis inhibitors. As the results in Fig. 4 show, cells in whichnew RNA synthesis was stopped by the addition of actinomycinD alone showed a rapid decline in the number of N-myctranscripts. Approximately one-half of the total N-myc RNAwas lost after 30 min of inhibition of new RNA synthesis. Y79cells treated with anisomycin without actinomycin D increasedtheir levels of N-myc RNA by over 2-fold in 90 min. Whenactinomycin D was added together with anisomycin or aftercells were exposed to anisomycin for either 30 or 60 min, thelevels of N-myc mRNA declined much less rapidly than whenprotein synthesis was not inhibited. Similar results were obtained when actinomycin D was added to cells after exposureto other inhibitors of protein synthesis (70 /¿Mcycloheximideor 50 /¿Memetine) for up to 4 h. These results suggesting thatstabilization of N-myc mRNA against a normally rapid decayprocess is responsible for a lengthening of the lifetimes and fora net increase in the levels of this RNA in Y79 cells.

DISCUSSION

Our results show that the level of N-myc mRNA increasedin neuronal cells treated with inhibitor of protein synthesis.This effect appears to be due to posttranscriptional stabilizationof short-lived transcripts rather than to increased transcription

250

200-

ocO)

o

Eo.o

150-

9 A

• A

+ ANI

+ AD (30 min)

+ AD (60 min)

1DO

\ —̂

-A

»•

—I"

30 60 90

min after ANI additionFig. 4. N-myc RNA levels in Y79 cells treated with protein and RNA synthesis

inhibitors. Anisomycin (ANI; 70 ¿IM)was added to Y79 cells at 0 time, and cellswere harvested for cytoplasmic RNA at 30-min intervals. Other cells were treatedwith 5 (i¡!/nilactinomycin D alone for 30 or 60 min. Also, actinomycin D wasadded to cells for 30 or 60 min (A) which had been treated with anisomycin at 0time.

of this gene. This stabilization was not a general phenomenonfor all mRNA in Y79 cells and may reflect the amplified stateof the N-myc gene in these cells. Likewise, Sive et al. (25)showed that protein synthesis inhibition superinduced histonemRNA in HeLa cells, but not /3-actin or heat shock mRNA.However, a number of transcripts show similar patterns ofposttranscriptional regulation in specific cell types. In ourstudy, HL-60 cells (which have amplified c-myc) showed a largeincrease in c-myc mRNA levels with protein synthesis inhibitionsimilar to the changes seen in c-myc mRNA levels in BALB3T3 cells (26). Other examples of genes with transcripts with

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REGULATION OF N-myc mRNA STABILITY

short lifetimes which can be prolonged by protein synthesisinhibition include c-fos in murine 3T3 cells (16), histone (27),glyceraldehyde-3-phosphate dehydrogenase in rat tissues (28),and c-myc and P53 expression during F9 cell differentiation(29).

Thus, N-wyc mRNA in neuronal cells is one of a large groupof genes the transcript levels of which can be rapidly elevatedby inhibiting a posttranscriptional regulatory mechanism. Shawand Kamen (30) have recently shown that the insertion of anAU-rich sequence into the 3'-untranslated region of a gene

encoding a normally long-lived mRNA caused the resultingtranscript to be very unstable in cells. This sequence had noeffect on transcription rate, and the instability of the mRNAcould be partially overcome by treatment with cycloheximide.Genes which normally produce transcripts with similar AU-rich sequences in 3'-untranslated regions include colony-stim

ulating factor, interferons, interleukins, and tumor necrosisfactor as well as the protooncogenes c-fos, c-sis, and c-myb (30).The human (1) and murine (31) N-myc sequences each haveuntranslated 3' regions which contain similar AU-rich se

quences. These sequences may be markers for linking themRNA to a rapid degradation mechanism. Mechanisms whichmay be responsible for regulating transcript stability includeRNases which recognize features of RNA which they specifically degrade (32) as well as translation-linked degradation of

certain mRNA (12).Although the level of N-myc expression in neuroblastoma

and retinoblastoma cells has been shown to be roughly proportional to the number of genomic sequences in a number of celllines (7), Lee et al. (8) have shown that primary neuroblastomatumors which did not contain amplified N-myc genes stillexpressed 4- to 5-fold higher N-myc RNA levels than did retinalcells. Also, Schwab et al. (7) showed that N-myc amplificationwas confined to the neuroblastoma tumor and was not presentin normal tissues of the same patient. These data suggest thatrelatively high levels of N-wyc expression (whether due toamplified genomic sequences or enhanced expression of a singlegene) correlates well with sustained growth and suppresseddifferentiation in neuronal cells. Furthermore, Thiele et al. (13)have shown that retinoic acid-induced differentiation of neuroblastoma cells in vitro was accompanied by a decrease in N-mycexpression; c-myc expression levels were not altered by retinoicacid treatment in this system. Similar effects on c-myc mRNAlevels have been observed with agents that modify differentiation in other systems (14, 15). Thus, the similarities betweenN-myc expression control and posttranscriptional expressionregulation of a number of other genes, particularly c-myc withwhich N-myc shares sequence and organizational similarities(1,31), suggests that these genes control their mRNA levels bythe same general process. How such a posttranscriptional mechanism operates to stabilize specific short-lived mRNA and whatroles N-w>r may play in neuronal cell functioning have yet tobe determined.

ACKNOWLEDGMENTSWe thank Wen-Hwa Lee for many useful discussions as well as for

providing us with many of the plasmids used in this study. We alsothank Hisao Yokota for help and discussions throughout the course ofthis project.

REFERENCES

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11. Seeger, R. C, Brodeur, G. M., Sather, H., Dalton, A., Siegal, S. E.,Wong,K. Y., and D. Hammond. Association of multiple copies of the N-myconcogene with rapid progression of neuroblastoma. N. Engl. J. Med., 313:1111-1116. 1985.

12. Linial, M.. Gunderson. N., and Groudine, M. Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science(Wash. DC), 230: 1126-1132, 1985.

13. Thiele, C. J., Reynolds, C. P., and Israel, M. A. Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of humanneuroblastoma. Nature (Lond.), 313: 404-406. 1985.

14. Grosso, L. E., and Pilot, H. C. Transcriptional regulation of c-myc duringchemically induced differentiation of HL-60 cells. Cancer Res. 45:847-850,1985.

15. Reitsma, P. H.. Rothberg, P. G., Astin, S. M., Trial, J., Bar-Shavit, Z., Hall,A.. Teitelbaum, S. L., and Kahn, A. J. Regulation of myc gene expression inHL-60 leukemia cells by a Vitamin D metabolite. Nature (Lond.) 306: 492-494, 1983.

16. Kohl, N. E.. Gee, C. E., and Alt, F. W. Activated expression of the N-m>'cgene in neuroblastomas and related tumors. Science (Wash. DC), 226:1335-1337, 1984.

17. Greenberg, M. E., and Ziff, E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature (Lond.), 311: 433-438, 1985.

18. Favaloro, J., Treisman. R., and Kamen, R. Transcription maps of polyomavirus-specific RNA: analysis by two-dimensional nuclease S, gene mapping.Methods Enzymol. 65: 718-749, 1980.

19. Maniatis, T., Fritsch, E., and Sambrook, J. Molecular Cloning. A LaboratoryManual. Cold Spring Harbor. NY: Cold Spring Harbor Laboratory, 1982.

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1987;47:6310-6314. Cancer Res   Christopher M. Amy and James C. Bartholomew  Neuroblastoma and Retinoblastoma Cells

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