[CANCER RESEARCH 49. MIO-.1916. July 15. I989|

Reversible Suppression of c-myc Expression in a Human Colon Carcinoma Line bythe Anticancer Agent /V-Methylformamide'

Devasis Chatterjee, Andrew Mendelsohn, Peter R. Shank, and Todd M. Savarese2

Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

ABSTRACT

The anticancer agent /Y-methylformamide (NMF), which at high concentrations (170 IHM) induces cultured DLD-1 Clone A human coloncarcinoma cells to increase their doubling times and lose their tumorigenicity in nude mice (Cordeiro, R. F., and Savarese, T. M. Cancer Res.,46: 1297-1305, 1986), suppresses the expression of the c-myc protooncogene in these cells in a dose- and time-dependent manner. Thissuppression involves an inhibition of c-myc transcription rather than anincreased degradation of c-myc inKN \. and is reversed if NMF isremoved from the culture medium. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene, which is thought to be constitutive, isrelatively unaffected by NMF treatment. The NMF-mediated decreasein c-myc expression may be associated with the ability of this agent toincrease the doubling time of these cells, but there is no direct temporallink between the loss of c-myc expression and the NMF-induced loss oftumorigenicity.

INTRODUCTION

The antineoplastic action of NMF,' an agent which has

recently undergone clinical trials (1-3), has been recognizedsince the early 1950s (4). Interest in this compound is based onthe findings that (a) at high (100-200 ITIM)concentrations,NMF or its congener DMF can induce terminal differentiationof certain murine (5) and human (6) leukemic cell lines, and (¿>)NMF (and DMF) has activity against human solid tumors innude mice model systems (7).

The ability of NMF and DMF to induce maturation in solidtumor lines has also been examined. Against certain culturedhuman colon carcinoma cell lines such as the DLD-1 and HCT-15 lines, high concentrations of DMF (up to 103 m\i) andNMF (up to 170 mivi) have been shown to cause increaseddoubling times, a loss of clonogenicity in soft agar, and a lossof tumorigenicity in nude mice (8-11). In addition, DMF hasbeen shown to increase the expression of a mucoprotein antigenassociated with normal colonie mucosa (9). Since all of theseactions are reversed upon drug removal (8-11), it is questionable whether these changes represent differentiation per se: instead these polar solvents have been described as causing theexpression of a "less malignant phenotype" in these cell lines

(8-11). The mechanisms by which NMF exerts these actionsare not well understood. Induction of terminal differentiationin leukemias by polar solvents including NMF may occurthrough a nonspecific mechanism, as it has been shown usingthe HL-60 human promyelocytic leukemic cell system that thematuration-inducing potency of a series of these compounds isbetter correlated with molecular weight than with chemical

Received 6/24/88; revised 3/28/89; accepted 4/21/89.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.

' This investigation was supported by USPHS Grants CA-38630. CA-20892,and Research Career Development Award CA-01241 (T. M. S.) awarded by theNational Cancer Institute. DHHS. Support was also provided by a grant fromthe Ocean State Charities Fund and a Predoctoral Fellowship Award from thePharmaceutical Manufacturers Association (D. C.).

' To whom requests for reprints should be addressed, at Box G, BrownUniversity. Providence, Rl 02912.

' The abbreviations used are: NMF, jV-methylformamide; DMF, A/'.A/-dimetn-ylformamide; SDS. sodium dodecyl sulfate: PDGF, platelet-derived growth factor.

structure (12). It has also been suggested that NMF may exertmaturation-like actions on human colon carcinoma cells byphysicochemically altering the viscosity of plasma membranes(13), or via metabolic perturbations, such as modulations ofnonprotein thiol levels (11). It is not known how the cellularactions of NMF result in both growth inhibition and suppression of the malignant phenotype in cultured human coloncarcinoma cell lines. One hypothesis is that NMF alters theexpression of certain critical proto-oncogenes that may be involved in growth and/or maintaining the transformed phenotype in these cells (for reviews on the role of proto-oncogenesin these processes, see Refs. 14-16). One of the most widelystudied proto-oncogenes is c-myc, the cellular homologue of agene carried by the avian myelocytomatosis virus. The c-mycgene is involved in mediating the development of avian leukosisvirus-induced bursal lymphomas ( 17). Several studies have demonstrated that the expression of c-myc plays a pivotal role inthe regulation of mammalian cell growth and differentiation; c-myc is able to immortalize murine fibroblasts (18), transformimmortalized rodent and human fibroblasts (19), and is rapidlyand dramatically induced in mitogen-stimulated quiescentmouse fibroblasts and resting human lymphoid cells (20), aswell as in regenerating rat liver (21). In the process of differentiation-induction, a decline of c-myc expression is in manysystems an early event. For example, decreased levels of c-myctranscript are observed upon treatment of HL-60 leukemia cellswith maturation-inducing agents, including retinoic acid, dimethyl sulfoxide, and 1,25-dihydroxyvitamin D, (22-24).Chemical induction of differentiation of the Friend murineerthyroleukemia cell line results in a biphasic decrease in c-mycexpression (25, 26); differentiation induction can be blocked,however, if these cells are transfected with plasmids containingthe c-myc gene (27, 28). Given the important role that c-mycplays in both cell growth and differentiation in a number of celltypes, it was examined if alterations in this gene were involvedin the cytostatic and phenotype-changing actions of NMF onDLD-1 Clone A human colon carcinoma cells.

MATERIALS AND METHODS

Cell Culture. DLD-1 Clone A human colon carcinoma cells werecultured in RPM1 1640 media supplemented with 10% fetal calf serum,antibiotics, and buffers in a humidified incubator of 95% air, 5% CO2at 37°Cas previously described (8). In all experiments, DLD-1 CloneA cells (5 x IO5)were initially plated in 100-mm tissue culture dishes(Falcon plastics) in the above-mentioned media. After 24 h of incubation the medium was removed, cells washed twice in physiologicalsaline, and drug-free or drug-containing medium was added. Afterappropriate incubation times, cells were harvested with trypsin-EDTA,washed twice in Dulbecco's phosphate buffered saline, and stored at-80°Cfor RNA isolation.

RNA Isolation and Northern Blot Analysis. Total cellular RNA wasisolated using the guanidinium thiocyanate method with cesium chloride modificai ion (29, 30). RNA was quantitated by absorbance at 260nm and by comparison with known concentrations of yeast RNAstandards which were electrophoresed through an agarose gel andstained with ethidium bromide. Total cellular RNA was electrophoresedthrough a 1% agarose:6% formaldehyde gel at 70 V for 8 h (31). RNA

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NMF SUPPRESSION OF c-myc

was transferred onto Gene Screen filters (New England Nuclear, Boston, MA) for 20 h. The filters were air dried and baked in vacuo for 4h at 80°C.Prehybridization was done at 42°Cfor 16 h in a solution

containing 50% formamide (deionized), 0.2% polyvinyl-pyrrolidone,0.2% bovine serum albumin, 0.2% ficoll, 0.05 M Tris-HCl (pH 7.5),

1.0 M NaCl, 0.1% sodium pyrophosphate, 1.0% SDS, 10% dextransulfate, and denatured salmon sperm DNA (100 ^g/ml). Hybridizationwas performed at 42°Cfor 24 h in the same solution containing IO6cpm/ml of DNA probes labeled by nick-translation (32) with [a-':P]-

dATP. The Clal-EcoR\ fragment (1.8 kilobases) of the c-myc gene,corresponding to the third exon, was used as a probe (purchased fromOncor, Gaithersburg, MD). Filters were washed (as recommended bythe filter manufacturer) under low stringency at 57"C and exposed topreflashed Kodak XAR film with intensifying screens at -80°C.Filters

were also probed with the Pstl-Rsa\ fragment of HcGAP3, a partialcDNA clone representing 0.7 kilobase of the human glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) (kindly provided by Dr. RayWu, Cornell University). In cases where a filter was hybridized withmore than one probe, the initial hybridized probe was removed fromthe filters by incubation at 65°Cin 0.005 M Tris-HCl, 0.0002 M

NaiEDTA, 0.05% sodium pyrophosphate, 0.002% polyvinyl-pyrroli

done, 0.002% bovine serum albumin, and 0.002% ficoll for 2 h asrecommended by the filter manufacturer. "P-end-labeled H¡nd\\\ frag

ments of phage X DNA were included in each gel to serve as sizemarkers. Densitometry was used to quantify gene expression followingautoradiography on preflashed film.

Assay of Transcription Rates. The measurement of transcription ratesin DLD-1 Clone A cells was modified from various published reports(33-35). Nuclei from DLD-1 Clone A cells were isolated from cellstreated for 96 h with 170 HIM NMF and untreated cells. Harvestedcells were washed with phosphate-buffered saline and pelleted at 500 xg for 5 min. The cell pellet was resuspended in buffer containing 5 IHMMgCl2, 1 mM CaCI3, 20 IHMTris-HCl (pH 7.6), 14 HIMKCI, 14 mivi(3-mercaptoethanot, 20% glycerol, and 0.3% (v/v) Nonidet P-40 (NP-40), homogenized in a Dounce homogenizer, and centrifuged at 1000x g for 5 min at 4°C.The pellet was washed and nuclei were resuspended

in a buffer containing 25% glycerol, 36 mM 4-(2-hydroxylethyl)-l-piperazineethanesulfonic acid (pH 7.6), 3 mM MgCI:, 7 mM dithiothre-itol, and 170 mM KCI at a density of IO7 nuclei/ml. Nuclei (5 x 10")

from each experimental group were utilized for transcription assays.The resuspended nuclei were combined with 1 mM each of ATP, GTP,and CTP, 25% glycerol, 25 mM 4-(2-hydroxylethyl)-l-piperazineeth-anesulfonic acid (pH 7.6), 2 mM MgCI2, 2 mM MnCl?, 5.5 mM dithio-threitol, and 134 mM KCI. The reaction mixture was incubated for 25min at 26°Cwith 100 ¿iCi/mlof |«-'-P]UTP, and the reaction termi

nated by the addition of RNase-free DNase (100 units/ml). As anadditional control, the inhibitor of RNA polymerase II, «-amanitin,was added (2 Mg/ml) to reaction mixtures to demonstrate that theobserved transcriptional activity was RNA polymerase II dependent.After the transcription reaction, the nuclei were incubated at 37°Cfor

l h in a buffer containing 1% SDS, 5 mM EDTA, 10 mM Tris-HCl

(pH 7.4), and 200 ¿¿gof proteinase K. The reaction mixture wasextracted twice with an equal volume of phenolxhloroform and precipitated by the addition of yeast transfer RNA (10 *ig/ml), 200 mM NaCl,and 2.5 volumes of ethanol. The labeled pellet was resuspended in ahybridization solution consisting of 10 mM TES (pH 7.4), 0.5 mg yeasttransfer RNA/ml, 0.2% ficoll, 0.2% polyvinyl-pyrrolidone, 1% sodium

pyrophosphate, and 0.4% SDS. Nitrocellulose filters were prepared byimmobilizing 10 ng of DNA from the Xhol-EcoR\ fragment representing exons 2 and 3 of the c-myc gene (36), GAPDH, and plasmid PBR322 with a Bio Rad dot-blot manifold as suggested by the manufacturerand prehybridized for 4 h at 65°Cin the hybridization solution listedabove. Labeled transcription products were added (5 x IO6 cpm/ml)and the mixture hybridized at 65°C.The filters were washed with

several changes of 2x SSC (Ix SSC = 0.15 M NaCl, 0.0125 M sodiumcitrate, pH 7.0) at 65°C.The filters were air dried, and exposed toKodak X-Omat X-ray film at —¿�80°C.Results were quantitated by

excising spots from the filter and counting them by liquid-scintillation

spectrometry.

Tumorigenicity Studies. Tumorigenicity studies were carried out asdescribed previously (11).

RESULTS

NMF-mediated Suppression of c-myc Expression. Initial studies examined whether 170 mM NMF, a concentration previously determined to inhibit cell growth and suppress the malignant phenotype of DLD-1 Clone A human colon carcinomacells (11) could alter c-myc expression in these cells. DLD-1Clone A human colon carcinoma cells were continuously exposed to this concentration of NMF for up to 2 weeks (two cellpassages). At time intervals ranging from 24 h to 2 weeks afterthe addition of NMF, total cellular RNA was isolated andanalyzed for the expression of c-myc by Northern blot analysis.Fig. 1 shows that c-myc expression was reduced by approximately 22% compared to untreated control cells within 24 hfollowing exposure to NMF. Expression of c-myc decreasedthroughout the time interval tested with greater than 90%reduction in c-myc mRNA after 7 days of NMF treatment ascompared to untreated control cells. In a separate experiment,it was determined that c-myc expression remains at this depressed level (<10% of controls) if DLD-1 Clone A cells aretreated for up to four cell passages (4 weeks) with mediumcontaining 170 mM NMF (see Fig. 4 and Ref. 37). The samefilter was also probed with ':P nick-translated sequences coding

for part of the human glyceraldehyde 3-phosphate dehydrogenase gene, GAPDH (Fig. \B). Previous studies indicated thatthe GAPDH gene is expressed constitutively in several humantissues (38), and therefore serves as a control for amounts ofRNA and for general changes in gene expression induced byNMF. Densitometric analysis revealed that the level of GAPDHmRNA expressed in NMF-treated cells remains relativelysteady over the 2-week period of study. This suggests that NMFtreatment does not result in a generalized suppression of gene

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B1.4* •¿�§••*•

Fig. 1. Time course of the A'-methylformamide-induced decrease in c-mycexpression in DLD-1 Clone A cells. A, effect of NMF on c-myc expression inDLD-1 Clone A cells. DLD-1 Clone A cells (5 X lOVlOO-mm culture dish) wereplated in serum-supplemented RPMI 1640 medium as described in "Materialsand Methods." The medium was removed after 24 h. the cells washed in physio

logical saline, and fresh medium containing 170 mM NMF added. Cells werereplated in NMF-containing medium 7 days after initiation of drug treatment,and were maintained in drug-containing medium for a total of 14 days. At varioustimes, cells were harvested and total cellular RNA was isolated as described in"Materials and Methods." Ten fig of total RNA from each time interval was

electrophoresed through a 1% agarose. 6% formaldehyde gel and transferred ontoGene Screen membranes for 24 h. Filters were hybridized at 42"C for 24 h witha 32P-labeled c-myc probe (purchased from Oncor) (1 x 10*cpm/ml) as describedin the text. Lane 1. c-myc expression in logarithmically growing untreated controlcells; lanes 2-6, c-myc expression in cells exposed to 170 m\i NMF for thefollowing time periods: 24 h (lane 2). 48 h (lane 3). 96 h (lane 4), 1 days (lane5), and 14 days (lane 6). Kodak XAR film was exposed to the filter at —¿�80°Cfor96 h prior to development. B, effect of NMF on GAPDH expression in DLD-1Clone A cells. The same filter in A was stripped of c-myc probe as recommendedby the filter manufacturer and hybridized under the same conditions as describedabove with 1x10* cpm/ml of "P-labeled HcGAP.1 (a partial sequence of the

human GAPDH gene) probe. Filters were exposed to Kodak XAR film for 96 hat —¿�80eC.Transcript sizes for all Northern blot analyses are given in kilobases as

determined from A lliml\\\ fragment markers.

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NMF SUPPRESSION OF c-myc

expression in these cells. The NMF-mediated decrease in c-myc expression in these cells was also dose-dependent over aconcentration range of 42.5-170 mM (Fig. 2).

We then examined whether the time course of the NMF-induced loss of the malignant phenotype in these cells paralleledthe time course of the NMF-mediated decrease of c-myc expression. For any given cell type, the only true indicator of malignancy is tumorigenicity in an appropriate host (39, 40). Therefore, cultured DLD-1 Clone A cells were incubated in mediumcontaining 170 mM NMF, and at various intervals cells wereharvested and injected s.c. into the flanks of nude mice (IO6

cells injected/mouse). After 10 weeks, the incidence of tumorswas assessed. As shown in Table 1, at least 21 days of exposureto NMF is required before these cells display a major loss oftumorigenicity; 28 days of exposure is needed to produce acomplete loss of tumorigenicity. Since the nadir of c-mycexpression occurs after approximately 7 days of treatment withNMF (Fig. 1), these data indicate that there is no direct temporal association between loss of c-myc expression and loss oftumorigenicity in NMF-treated DLD-1 Clone A cells.

Previous studies have shown that the actions of NMF and itscongener, yV,/V-dimethylformamide, on cultured human coloncarcinoma cells, including growth inhibition and decreasedclonogenicity in soft agar are reversed upon their removal from

12345

B

2.3*

1.4*Fig. 2. Dose-dependent loss of c-myc expression in DLD-1 Clone A cells

treated with NMF. A, effect of increasing doses of NMF on c-myc expression.DLD-1 Clone A cells were plated as described in Fig. 1. After 24 h the mediumwas removed, cells washed in physiological saline, and fresh medium addedcontaining either no addition (lane /), 42.5 mM NMF (lane 2), 85 mvi NMF(lane 3). 127.5 m\t NMF (lane 4), and 170 mM NMF (lane 5). Cells wereharvested after 7 days and total cellular RNA isolated as described in "Materialand Methods." Twenty »jgof total RNA from each sample were utilized forNorthern blot analysis to examine c-myc expression. Filters were exposed toKodak XAR film for 96 h at -80°C. B, effect of increasing doses of NMF on

GAPDH expression. The filter in A was stripped and reprobed with labeledHcGAPJ as described in Fig. 1. The filter was exposed to Kodak XAR film for96 h at —¿�80"C.Transcript sizes are given in kilobases as determined from X

Hindlll fragment markers.

Table 1 Time course of the decrease in tumorigenicity of DLD-1 Clone A humancolon carcinoma cells treated with NMF

DLD-1 Clone A cells (5 x lO'/lOO-mm tissue culture dish) were plated inRPMI 1640 medium as described in "Materials and Methods." After 24 h of

incubation, the medium was removed, the cells washed in physiological saline,and fresh medium added containing 170 mM NMF. Cells were harvested at timeintervals ranging from 7 days to 28 days after drug addition. At each time point,cells were washed four times in physiological saline, and measured for viabilityby trypan blue exclusion. Athymic nude mice bearing the nu/nu gene on a Swissbackground were obtained from the Roger Williams Cancer Center Animal CareFacility (Providence, RI). Mice were randomized into groups of 8-10 animalsand each mouse injected s.c. with 10' cells that had been treated for the appro

priate time interval. Tumor incidence was assessed, 10 weeks after inoculation,as the number of mice in which palpable tumors (>100 mm3) developed versus

the total number of mice given injections.

Length of treatmentwith 170mMNMFControl

(no treatment)7 Days

14 Days21 Days28 Days%

viability attime of inoculation91

87848889Incidence

of tumors,10-week

postinjection10/10

9/98/96/100/10

the culture medium (8-11). Consequently, we examinedwhether c-myc expression and growth in DLD-1 Clone A cellswould return to control levels if NMF was removed from themedium. Cells were treated for 96 h (4 days) with 170 mMNMF to achieve suppression of c-myc expression, then themedium was removed, the cells washed twice in physiologicalsaline, and drug-free medium was added. As a control, somedishes received fresh medium containing 170 HIMNMF following the initial 96 h of NMF treatment. Cells were then harvestedat 0, 24, and 96 h after the addition of fresh NMF-free orNMF-containing media, and total cellular RNA was isolatedand analyzed for c-myc expression. Fig. 3 shows that after theinitial 96-h exposure to NMF, the level of c-myc mRNA wasdecreased relative to untreated control cells (lane 2 versus lane1). However, c-myc expression is nearly restored to controllevels 24 h following the removal of NMF (lane 4). In contrast,if the cells were continuously exposed to 170 mM NMF for anadditional 96 h, after the initial 96-h treatment period, thelevels of c-myc continued to remain suppressed (lane 5). In aparallel study, labeled thymidine incorporation was inhibited63% after 96 h of treatment of NMF as compared to untreatedcontrol cells, but returned to levels 9% below control cells 96 hafter the removal of NMF from the medium (Table 2). Theseexperiments demonstrate that the NMF-mediated suppressionof c-myc expression and growth depends on the presence of thedrug and is reversible, at least after a 96-h drug-exposure period.

However, since these cells require 28 days of exposure toNMF to lose their tumorigenic potential (Table 1), it was morerelevant to examine the reversibility of NMF-mediated suppression of c-myc expression at this time period. Once again, asignificant recovery of c-myc expression was observed within96 h after removal of NMF from the culture medium (Fig. 4).Likewise, the incorporation of thymidine in DLD-1 Clone Acells that had been exposed to NMF for 28 days was inhibited76% when compared to untreated control cells, and was restored to 32% below controls 96 h after the removal of the drugfrom the medium. Thus the NMF-mediated loss of c-mycexpression is reversible after 4 days of drug treatment, whenthere is no decrease in tumorigenicity, and after 28 days of drug

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B1.4»-

Fig. 3. Reversal of the NMF-mediated loss of c-myc expression in DLD-1Clone A cells upon drug removal (after 96 h of drug exposure). A, effect ofremoval of NMF on c-myc expression. DLD-1 Clone A cells were plated asdescribed in Fig. 1. After 24 h the medium was removed, cells washed twice inphysiological saline and medium containing 170 mM NMF added. Cells wereexposed to NMF for 96 h, after which the medium was removed, and cells washedtwice in saline. These cells then received either drug-free medium (Group 1) ormedium containing 170 mM NMF (Group 2). Group 1 cells were harvested atvarious times after addition of drug-free medium; 0 h (lane 2), 24 h (lane 4), and96 h (lane 6). Group 2 cells, which underwent continuous exposure to NMF,were harvested 24 h (lane 3), and 96 h (lane 5) after the addition of fresh NMF-containing medium. Lane I, logarithmically growing untreated Clone A cells.Total cellular RNA was isolated and 20 ^g from each sample was examined forc-myc expression using Northern blot analysis as described in Fig. 1. Filters wereexposed to Kodak XAR film for 96 h at -80'. B, effect of removal of NMF on

GAPDH expression. The filter from panel A was stripped and hybridized with theGAPDH probe as described in Fig. 1. Kodak XAR film was developed after 96-hexposure.

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NMF SUPPRESSION OF c-myc

Table 2 Reversal of the NMF-mediated inhibition of/'H/lhymidineincorporation into DLD-1 Clone A human colon carcinoma cells

DLD-1 Clone A cells (5 x 10") were plated in 60-mm tissue culture dishes.

After 24 h (Day 0), the medium was removed and fresh medium added containingeither 170 mM NMF or no addition. After 4 or 28 days of incubation, the mediumwas removed, cells washed twice with 0.9% saline, and drug-free medium addedto all dishes. The plates were then incubated up to an additional 96 h (Day 8 andDay 32). At the indicated times during these manipulations. 5 fiCi/ml [*H]-thymidine (91 Ci/mmol) was added to the dishes; after l h of incubation at 37"C.incorporation was stopped by the addition of 10% ice-cold trichloroacetic acid.Precipitated cell material was washed several times in 10% trichloroacetic acid,and these pellets dissolved in 0.5 N NaOH for 15 min at 80°C.Neutralized

samples were added to 10 ml Aguasol scintillant and counted.

Days oftreatment4

5 (24 h after the re-cpm/IO'

cells"Control

NMF10,492

±811 8.689 ±9419.717 ±1,742 3.514 ±458

10.841 ±2,104 4,553 ±721%

inhibition17

6357

moval of NMF)8 (96 h after the removal of NMF)

2829 (24 h after the re

moval of NMF)32 (96 h after the re

moval of NMF)

11,266± 1.083 10.252 ±619

11.533 ±1.41112,110 ±744

10,049 ±823

2,752 ±3223.633 ±567

6.833 ±1,165

7669

32

' Values, mean ±SD of duplicate samples from two independent experiments.

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B1.4»

Fig. 4. Reversal of the NMF-mediated loss of c-myc expression in DLD-1Clone A cells upon drug removal (after 28 days of drug exposure). I. the sameexperiment was performed as described in Fig. 3 except that DLD-1 Clone Acells were exposed continuously to 170 m\t NMF for 28 days. After this treatmentperiod, NMF was removed, the cells divided into two groups and either drug-freemedium added (Group 1) or medium containing 170 mM NMF added (Group 2).Group 1 cells were harvested at 0 h (lane 2), 24 h (lane 4). and 96 h (lane 6) afterthe addition of drug-free medium. Cells in Group 2 remained exposed to 170 m\iNMF for an additional 24 h (lane 3) and 96 h (lane 5). Lane I. exponentiallygrowing DLD-1 Clone A cells. RNA was isolated from both groups. Northernblot analysis performed and the filters hybridized to c-myc DNA as described inFig. 3. B, the same filter was stripped and hybridized to GAPDH as previouslydescribed. Filters were exposed to Kodak XAR film for 96 h at -80°C.

treatment, when tumorigenic potential is completely abolished.This suggests that NMF's c-myc suppressing activity cannot byitself be correlated with NMF's ability to decrease the tumori

genic phenotype of these cells. Furthermore, these findingsdemonstrate that even a prolonged suppression of c-myc levels(over a period of 4 weeks), does not trigger this cell type todifferentiate to a postmitotic state.

Mechanistic Studies on NMF-mediated Suppression of c-mycExpression. NMF may decrease steady state levels of c-myctranscripts in DLD-1 Clone A cells by (a) decreasing the rateof c-myc transcription, and/or (b) decreasing the stability of thec-myc mRNA. To investigate the effect of NMF on the stabilityof the c-myc mRNA, DLD-1 Clone A cells were grown in thepresence or absence of 170 mM NMF for 96 h as previouslydescribed, the medium was removed and fresh medium addedcontaining 5 ng/m\ of the transcription inhibitor, actinomycinD. Cells were then harvested at various time intervals rangingfrom 30 to 120 min, and total cellular RNA isolated forNorthern blot analysis. The half-life of c-myc mRNA in NMF-treated cells (37 min) was comparable to that in untreated

control cells (28 min) (Fig. 5). Treatment with NMF did notsignificantly alter the half-life oíGAPDH mRNA (115 min) ascompared to untreated control cells ( 125 min) (data not shown).Therefore, NMF does not exert its effect on the expression ofc-myc in DLD-1 Clone A cells at the level of mRNA stability.

It was then examined whether NMF affects the rate of c-myctranscription in DLD-1 Clone A cells. Preliminary studiesdetermined that a linear response for the rate of transcriptionof c-myc in DLD-1 Clone A cells is obtained if 5 x 10" cellnuclei are incubated for 25 min at 26°C.Nuclei were isolated

from untreated DLD-1 Clone A cells and cells that had beentreated for 96 h with 170 mM NMF. The rate of c-myc transcription in nuclei from NMF-treated cells was reduced 41%compared to that in untreated control cells (Fig. 6). Thus,inhibition of transcription can apparently account in part forthe decrease in the steady state level of c-myc transcripts thatis observed after 96 h of NMF treatment (Fig. 3). The NMF-

0-<J

60

TIME(min)

IZO

Fig. 5. Effect of NMF treatment on the stability of c-myc mRNA in DI.D-IClone A cells. DLD-1 Clone A cells were plated as described in Fig. 1. After 24h. the medium was removed, the cells were washed in saline, and drug-containingor drug-free medium was added. Cells were incubated for 96 h, at which time themedium was removed and fresh medium added containing 5 ..i1,ml of actinomycinD. Cells were then harvested at 30. 60. 90. and 120 min after initiation ofactinomycin D treatment. DLD-1 Clone A cells that did not receive actinomycinD were harvested after 96 h (0 time). Total cellular RN A was isolated as previouslydescribed and 20 jig of total RNA from each sample was utilized for Northernblot analysis. Filters were exposed to Kodak XAR film for 36 h at —¿�80*C.Half-lives from control (O) and NMF-treated cells (•)were quantitated as follows:radiolabclcd bands were excised from the niters, placed into vials containing 5ml HiO, and counted.

CON NMF

c-myc

(X -AMANITIN

GAPDH

PBR 322Fig. 6. Effect of NMF treatment on the rate of c-myc transcription in DLD-1

Clone A cells. Nuclei were isolated from untreated DLD-1 Clone A cells (CON)or DLD-1 Clone A cells treated for 96 h with 170 mM NMF (NMF). Nuclei (5x IO6) from each experimental group were incubated in transcription reactionsfor 25 min at 26"C, then the reactions were stopped, labeled RNA isolated, andhybridized onto nitrocellulose filters containing 10 pg of (a) the Xhol-EcoRlfragment of c-myc representing exons 2 and 3, (b) GAPDH, or (c) pBR 322 in aBio-Rad dot-blot manifold. The filters were washed as described in "Materialsand Methods." Samples used to determine the inhibition of RNA polymcrase IIby n-amanitin were hybridized to 5 vg of c-myc DNA. Filters were exposed toKodak XAR film for 24 h at -80"C. Results were quantitated by excising the

labeled spots from the filters and counting them in a liquid scintillation counteras described in Fig. 5.

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NMF SUPPRESSION OF c-myc

mediated reduction of c-myc transcription rate was relativelyspecific as there was no significant change in the rate of transcription ofGAPDH (Fig. 6).

DISCUSSION

These studies demonstrate that the experimental anticancerdrug, NMF, suppresses the expression of the pivotal protooncogene, c-myc, in the DLD-1 Clone A human colon carcinoma line. Major findings include: (a) this suppression is reversed upon removal of NMF from the culture medium, (b)this suppression involves an inhibition of c-myc transcriptionrather than an increased degradation of c-myc mRNA, and (c)the NMF-mediated decrease in c-myc expression can be associated with this agent's growth-inhibitory effect, but not withthis compound's ability to induce these cells to lose their

tumorigenic potential in nude mice.How NMF inhibits c-myc transcription is unknown; part of

the problem is that the factors which normally regulate c-myctranscription have not been elucidated. In fibroblast systems,ligands that activate protein kinase C directly, e.g., 12-0-tetra-decanoylphorbol 13-acetate, or indirectly, such as PDGF andbradykinin, which elevate the production of the endogenousprotein kinase C activator, diacylglycerol, via a receptor-mediated turnover of membrane phosphoinositides, have beenshown to induce c-myc expression (20, 34, 41, 42). Proteinkinase C in turn may activate one or more of the so-calledtranscription-activator factors, which can enter the nucleus andbind to regulatory elements of particular genes thereby alteringtheir expression (for reviews on these protein factors, see Refs.43 and 44). At least two such phorbol ester-responsive factors,designated AP-1 and AP-2, have been characterized (45-47); itis noteworthy that AP-2 is capable of binding to the human c-myc promoter (47). Hypothetically, NMF (or a metabolite) maybe interfering with the transduction of c-myc-inducing signalssomewhere along this pathway, resulting in a decrease in therate of c-myc transcription. We are currently screening variousgrowth factors and regulatory subtances for their ability tomodulate c-myc levels in human colon carcinoma lines so thatthis postulate may be tested.

Other hypotheses involve two recently described mechanismsthat may play an inhibitory role in the regulation of c-mycexpression, i.e., "antisense" transcription and transcription at

tenuation of c-myc. It is now known that c-myc can be transcribed in the "antisense" direction (48). While the physiologi

cal significance of this phenomenon is not understood, it isknown that introduction of vectors designed to express anti-sense c-myc into particular leukemic cell lines results in adecrease in endogenous c-myc transcript levels, inhibits cellproliferation, and induces differentiation (49, 50). Also, Bentleyand Groudine have reported that transcription of c-myc isattenuated (i.e., there is a block of elongation) between exons 1and 2 during the differentiation of HL-60 human promyelocyticleukemia cells (51). The latter phenomenon may explain thediscrepancy between the amount of inhibition of c-myc transcription observed after 96 h of NMF exposure (41%; Fig. 6)and the decrease in the steady state level of c-myc expressionat this time interval (62%; Fig. 1, lane 4). If NMF were causingattenuation at a site within or downstream of exon 2, thesepartial transcripts would be detected in the transcriptionalassay, which employed a template containing exons 2 and 3,but not in the Northern blots which were probed with only exon3 of c-myc. In any case its conceivable that the inhibition of c-myc transcription effected by NMF may be through an activa

tion of these negative regulatory mechanisms.Another consideration is based on the finding that the mer-

capturic acid derivative of NMF, yV-(methylcarbamoyl)-yV-ace-tylcysteine is present in the urine of patients treated with thisagent; this indicates that NMF is converted to a carbamoylatingagent, possibly methylisocyanate (52). Isocyanates, which havebeen studied as nitrosourea breakdown products, have beenshown to interfere with RNA processing, DNA polymeraseactivity, and other aspects of nucleic acid metabolism (53, 54).This hypothesis may explain why the sulfhydryl-containingamino acid, 1-cysteine, which could act as a nucleophilic scavenger for carbamoylating agents and/or maintain reduced glu-tathione pools, partially ameliorates the NMF-mediatedsuppression of c-myc (37). However, it does not account for thefinding that NMF inhibits c-myc transcription relatively specifically, i.e., GAPDH transcription is unaltered by NMF treatment (Fig. 6).

There are several indications that the NMF-mediatedsuppression of c-myc expression and its growth-inhibitory action of this compound in the DLD-1 Clone A cell line areassociated. Both the dose response and the time course for thesuppression of c-myc parallels those for NMF's growth-inhibi

tory action (Table 2 and Ref. 11). Also, both c-myc levels andlabeled thymidine incorporation recover with a similar timecourse when NMF is removed (Table 2). The role of c-myc incell growth is best understood in immortalized, nontransformedmurine fibroblast lines, e.g., BALB/C-3T3 cells. Quiescent cellsof this type can be made "competent" to enter the cell cycle byc-wyc-inducing polypeptide growth factors, including fibroblastgrowth factor and PDGF (20, 55). c-Myc is expressed at approximately equivalent levels throughout all phases of the cellcycle suggesting that c-myc may be necessary for progressionthrough the cell cycle, in addition to exiting from G0 (56, 57).Application of this model to the colon carcinoma cells studiedhere must be done with caution, since c-myc expression andgrowth competency in transformed cells can be independent ofserum growth factors (58). Nevertheless, an NMF-mediatedsuppression of c-myc expression might result in decreasedgrowth competency, making progression through the cell cyclemore difficult. Indeed, NMF (170 HIM) has been shown toincrease the doubling time of DLD-1 Clone A cells by approximately twofold (11). The component of the cell cycle effectedby the yv-alkylformamides is controversial: Leith et al. (59) havereported that NMF treatment prolongs all phases of the cellcycle equally in the DLD-1 system, whereas Bill et al. (60) haveobserved that NMF-treated TLX-5 murine lymphoma cellsaccumulate in the Gìphase of the cell cycle. In any case, it maybe difficult to establish a direct cause-and-effect relationshipbetween changes in c-myc levels and changes in cellular doubling time. In human fibroblasts, the c-wyc-inducing effect ofPDGF can be separated from its mitogenic action (42), and c-myc can be elevated by epidermal growth factor in A431 epidermal carcinoma cells without altering proliferation (61). IfDLD-1 Clone A cells whose c-myc gene copy number (and c-myc expression) is increased by transfection with a c-mycexpression vector display increased growth rates and are relatively insensitive to the growth-inhibitory actions of NMF, theargument that an NMF-mediated suppression of this proto-oncogene results in an increased doubling time would bestrengthened.

Can the suppression of c-myc by NMF account for thiscompound's ability to inhibit the expression of the malignant

phenotype in this cell type? In several other solid tumor systemsa decline of c-myc levels precedes terminal differentiation, for

3914

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NMF SUPPRESSION OF c-myc

example, in the induction of differentiation of the F9 teratocar-cinoma line by retinoic acid and cyclic AMP (62, 63). Also, adecrease in the expression of N-myc, which shares limitedhomology with c-myc, precedes growth arrest and differentiation of SMS-KCNR neuroblastoma cells (64). Reduced expression of the c-myc proto-oncogene accompanies induction ofdifferentiation of certain malignant cell lines of hematopoieticorigin, e.g., HL-60 promyelocytic leukemia and Friend eryth-roleukemia (MEL) cells, by chemical agents such as dimethylsulfoxide (23, 24, 26, 65). However, a decrease in c-myc isneither sufficient nor obligatory for MEL erythroid differentiation (66). In the present study, there does not appear to be adirect temporal link between the NMF-mediated loss of c-mycexpression and the loss of the tumorigenicity of DLD-1 CloneA cells. Furthermore, the NMF effect on c-myc is reversibleeven after 4 weeks of drug exposure, ruling out the possibilitythat long-term treatment with this agent induces a permanentloss of the cell's ability to express c-myc, leading to differentia

tion and a loss of tumorigenic potential. Nevertheless, it cannotbe ruled out that a prolonged c-myc suppression could lead toadaptations in the expression of other crucial genes that resultin a loss of the malignant phenotype in these cells.

Finally, the ability of NMF to suppress c-myc in coloncarcinomas may be of chemotherapeutic importance. The levelof c-myc expression in primary human colon carcinomas hasbeen reported to be considerably elevated (5-40-fold) relativeto that in normal colonie mucosa (67, 68); this elevation doesnot involve gene amplification or rearrangement (67). Thusagents which inhibit c-myc expression may have a relativelyselective effect on tumor tissue. Although NMF's toxicity has

limited its clinical usefulness (1,2,4), studies on its mechanismof action may lead to the design of more effective, less toxiccolon carcinoma-directed chemotherapeutic agents.

ACKNOWLEDGMENTS

The authors wish to thank Dr. John R. Coleman for the use of hislaboratory facilities for portions of this work and Dr. John D. Nagleand William Montigny for their advice on the transcription assays.

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-MethylformamideNCarcinoma Line by the Anticancer Agent Expression in a Human ColonmycReversible Suppression of c-

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