inhibition of proliferation by c-myb antisense oligodeoxynucleotides in colon adenocarcinoma cell...
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(CANCER RESEARCH 51, 2897-2901, June 1, 1991]
Inhibition of Proliferation by c-myb Antisense Oligodeoxynucleotides in ColonAdenocarcinoma Cell Lines That Express c-myb1
Cecilia Melani, Licia Rivoltini, Giorgio Parmiani, Bruno Calabretta,2 and Mario P. Colombo2
Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy [C. M., L. R., G, P., M. P. C.J, and Department of Pathology and Fels Institute for CancerResearch and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania [B. C.]
ABSTRACT
Steady-state mRNA levels of the protooncogene c-myb were measuredby Northern blot analysis in the human colon carcinoma cell lines LoVo,the doxorubicin-resistant derivative LoVo/Dx, Colo 205, and HT 29.Overexpression of c-myb mRNA was detected in the Colo 205 cell line,probably because of gene amplification, while in human HT 29 cells c-myb was not expressed at a detectable level. Comparison between LoVoand LoVo/Dx cell lines showed that c-myb mRNA levels were muchhigher in the doxorubicin-resistant derivative than in the parental line, c-myb antisense Oligodeoxynucleotides inhibited cell proliferation only inthe cell lines with detectable mRNA c-myb (LoVo, LoVo/DX, and Colo205). The dose of antisense exerting inhibitory effect was related to thelevels of c-myb mRNA expression. Inhibition of c-myb expression inantisense-treated LoVo/DX cells was demonstrated by the reverse tran-scriptase polymerase chain reaction technique.
LoVo/Dx cells were induced to differentiate by treatment with dimeth-ylformamide to determine whether down-regulation of c-myb expressionwould accompany the process of differentiation. During the treatmentwith dimethylformamide the expression of c-myb decreased in parallelwith the reduction of cell growth, while terminal differentiation of thesecells was associated with changes in the expression of carcinoembryonicantigen and laminili receptor genes.
Our findings demonstrate that the expression of c-myb is importantfor the proliferation of colon carcinoma cell lines and suggest that therole of this protooncogene is not restricted to cells of hematopoietic originbut is more general than previously thought.
INTRODUCTION
The protooncogene c-myb is preferentially expressed in he-mopoietic cells where it appears to play a key role in regulatingcell proliferation and, perhaps, differentiation. In normal hematopoietic cells, c-myb protein levels vary according to thelevel of differentiation, with very low levels of c-myb proteinbeing detected in terminally differentiated hematopoietic cells(1, 2). In the mouse Friend erythroleukemia cell line, F-MEL,constitutive expression of c-myb prevents terminal differentiation, while in human myelogenous leukemia cell lines inducedto differentiate, c-myb down-regulation is an early event thatprecedes differentiation (3-5).
In nonhematopoietic cells c-myb expression has been detected in small cell lung cancer (6), teratocarcinoma (7), neuroblastoma cell lines (8), primary colon tumors (9), and coloncarcinoma cell lines (10), suggesting that c-myb expression isnot strictly tissue specific but might be associated with cellproliferation in tissues of different origin and might play a rolein early embryonal development (11). Although the associationof c-myb with cell proliferation has been demonstrated in many
Received 8/28/90; accepted 3/22/91.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 Associazione Italiana per la Ricerca sul Cancro.Milan, Italy, by NIH Grant CA4678, and by American Cancer Society GrantCH-455. B. C. is a Scholar of the Leukemia Society of America.
2To whom requests for reprints should be addressed, at Department of
Pathology, School of Medicine, Temple University, Broad and Ontario Streets,Philadelphia, PA 19140 [B.C.]; or Division of Experimental Oncology D. InstitutoNazionale Tumori, Milan, Italy [M.P.C.].
cell populations, the exact role of this gene in proliferation anddifferentiation is not yet well established.
A useful tool to analyze the functional relevance of a geneproduct is to inhibit its expression by exposing target cells tosynthetic Oligodeoxynucleotides complementary to the genetranscript (12). This approach has been used to demonstratethe functional relevance of c-myb expression in the hemopoieticsystem (13-15). Normal hematopoietic colony formation andproliferation of myelogenous leukemia cell lines were bothaffected by exposure to c-myb AS3 Oligodeoxynucleotides to an
extent that depended on the proliferative capacity and level ofdifferentiation of the cell population tested (13-15).
To determine whether c-myb expression is also necessary forthe proliferation of epithelial neoplastic cells, we first comparedlevels of c-myb expression in four colon carcinoma cell lines,LoVo, its doxorubicin-resistant subline LoVo/Dx, Colo 205,and HT 29, with those in normal colonie mucosa. Next, weexposed neoplastic cells to c-myb AS Oligodeoxynucleotidesand found that inhibition of c-myb expression impairs coloncarcinoma cell lines proliferation. Analysis of c-myb expressionin the AS-treated LoVo/Dx cell line by the reverse transcriptasepolymerase chain reaction (PCR) technique revealed that c-mybmRNA levels were down-regulated by AS treatment. The impaired proliferation did not appear to be associated with celldifferentiation, as shown by the morphology of AS-treated cells.In LoVo/Dx cells induced to differentiate by treatment withDMF, differentiation was associated with growth arrest,changes in the cell phenotype, and variation in the expressionof c-myb, CEA, and one of the laminin receptor genes.
MATERIALS AND METHODS
Cell Culture and Oligodeoxynucleotide Treatment. LoVo, Colo 205,and HT 29 cell lines were all derived from human colon adenocarci-nomas (16). Doxorubicin resistance was induced in the LoVo/Dxsubline by prolonged culture in medium containing doxorubicin (17).Cells were cultured in Ham's F-12 medium (Gibco) supplemented with
10% fetal calf serum (Biological Industries), pretreated for 15 min at65°C,2 mM glutamine, and antibiotics (MA Bioproducts). Exponen
tially growing cells were detached by trypsin-EDTA (Flow) and used toisolate nucleic acids. For Oligodeoxynucleotide treatment, 3x10" cellswere seeded in 24-well plates (Costar) and c-myb S (5'-GCCCGAA-GACCCCGGCAC-3') or AS (5'-GTGCCGGGGTCTTCGGGC-3')
Oligodeoxynucleotides (codons 2-7) were added to the culture mediumfor 3 days at 40, 30, and 20 Mg/ml, respectively. In the dose-responseexperiments the cells were exposed to three different decreasing dosesof AS to obtain concentrations which were 'A, Vio,and '/«of the initial
dosage described above; S oligomers were only given at the highestdose. Control cells were cultured in the same conditions without Oligodeoxynucleotides. After 3 days cells were recovered by mechanicaldetachment, counted, and analyzed for viability by trypan blue dyeexclusion. To determine the cell proliferation rate, 3 x 10" S- and AS-
treated and untreated cells were washed after treatment with Oligodeoxynucleotides, pulsed with [3H]dThd (1 ¿iCi/well),and incubated for 6 h
3The abbreviations used are: AS, antisense: S, sense: SSC, sodium saline
citrate: SDS, sodium dodecyl sulfate; cDNA, complementary DNA; DMF, dimethylformamide; PCR, polymerase chain reaction; CEA, carcinoembryonicantigen; dThd, thymidine.
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c-myì>ANTISENSE OLIGODEOXYNUCLEOTIDES
at 37°C;cells were then washed and fixed on glass fiber dishes by using
a cell harvester apparatus (Titertek Cell Harvester 550, Flow Laboratories), and radioactivity was measured using a beta counter. Nonspecific radioactivity was measured in the culture medium and eliminatedfrom the counts. Differentiation was induced in LoVo/Dx cells byadding DMF to the medium at a final concentration of 0.8% for 15-25 days; during this period, cells were collected every 4-5 days untilfully differentiated and analyzed for gene expression and [3H]dThd
incorporation.Nucleic Acids Preparation and Hybridization. Simultaneous isolation
of RNA and DNA from colon carcinoma cell lines and fresh normalcolonie mucosa was performed by the guanidine isothiocyanate/cesiumchloride method (18). Cells were dissolved in 4 M guanidine isothio-cyanate-25 mM sodium citrate-0.5% Sarkosyl-0.7% /3-mercaptoethanol
by vortexing. Cell lysate was loaded on a cesium chloride cushion (4.95M CsCl-0.1 M sodium acetate-5 mM EDTA) and centrifuged for 16 hat 35,000 rpm at 25°C.DNA was recovered from the viscous interphase
of the cesium chloride gradient after ultracentrifugation and total RNAfrom the pellet at the bottom of the tube. DNA was treated withproteinase K (0.2 mg/ml), extracted twice with phenol/chloroform/isoamylalcohol (25:24:1), and ethanol precipitated; total RNA wasextracted twice with chloroform/isobutanol and ethanol precipitated.Total RNA (20 g) was denatured, separated on 1% agarose/formalde-
hyde gel and transferred to nylon filters (Hybond N, Amersham). ForSouthern blotting, 5 ^g of DNA was digested with the appropriaterestriction enzyme under conditions suggested by the manufacturer(Biolabs), electrophoresed on a 0.8% agarose gel, and transferred tonylon filters. Nucleic acids were bound to filters at 80°Cfor 2 h.
Prehybridization and hybridization were performed as described (19).Filters were washed twice in 2x SSC-0.1% SDS at room temperaturefor 15 min and then two or three times in 0.2x SSC-1.0% SDS at 52°C
for 30 min. To quantitate c-myb expression densitometric scanning of
the Northern blots was performed on a LKB Ultroscan XL.Reverse Transcription PCR. Total RNA was extracted from 3-8 x
IO5 LoVo/Dx cells treated with c-myb oligodeoxynucleotide as above.RNA was precipitated with 20 g of Escherichia coli tRNA and resus-pended in Ix reverse transcriptase buffer (BRL); 100 ng of 3' c-mybprimer was annealed at 55°Cfor 90 min and the first strand of cDNA
was synthesized using 400 units of MoMLV reverse transcriptase(BRL) at 37°Cfor 1 h. The reaction was stopped and the mixturediluted in Ix Tag polymerase buffer and 5' and 3' primers were added
to a final concentration of 2 ng/liter each, together with 2.5 units ofTaq polymerase (Cetus). Sixty cycles of PCR were performed in aPerkin Elmer thermal cycler by annealing at 55°C,synthesizing at72°C,and denaturing at 95°C.Positive and negative controls were used
to exclude amplification of contaminating sequences. The PCR productwas run on a 2% Nu Sieve agarose gel and transferred to a nylon filter(Hybond N, Amersham) by alkaline blotting. The filter was hybridizedat 50°Cto a synthetic oligonucleotide probe which was 5'-end labeled
with T4 polynucleotide kinase (Promega Biotech, Madison, WI) and7-32P-ATP; after hybridization filters were washed in 2x SSC/0.1%SDS at room temperature for 10 min and once at 50°Cfor 30 minbefore overnight exposure to X-ray films at 80°C.
Probes and Oligonucleotides. The human c-myb probe is a 0.6-kilobase long EcoRl fragment derived from the BCM-8 clone (20). Thelaminin receptor probe is a 280-base pair Aval/EcoRl fragment, 98%homologous to the COOH terminus of human cDNA (21)4; CEA-1 is
a 540-base pair Pstl fragment of cDNA which recognizes the humancarcinoembryonic antigen gene (22), and the histone H3 probe is a 2.1-
kilobase £coRl fragment from the pFO535 clone (23). All purifiedinserts were labeled at high specific activity with the random-primingmethod using a--12P-deoxycytidine (24). A radioactive probe, 2 x IO6
cpm, were used per ml of hybridization solution, c-myb S and ASoligodeoxynucleotides, 5' and 3' primers for PCR, and the synthetic
50-mer c-myb probe were made using a DNA synthesizer according to
the previously published sequences (15).
RESULTS
Expression of c-myb Oncogene in Colon Carcinoma-derivedCell Lines. Northern blot hybridization revealed that c-myb wasexpressed in both LoVo/Dx cell lines and overexpressed inColo 205, while HT 29 as well as normal colonie mucosa didnot show detectable levels of transcript at an exposure timewhich, in our study, was never longer than 6 days (Fig. 1). c-myb mRNA was of the expected size, although the transcriptresolved as a doublet of 3.8 and 2.5 kilobases in both untreatedand DMF-treated LoVo/Dx cells (Fig. 1).
Densitometric scanning of the Northern blot revealed that c-myb expression was 15-fold higher in LoVo/Dx than in LoVo.The greater abundance of c-myb mRNA in LoVo/Dx than inLoVo cells appeared to correlate with the higher proliferativeactivity of this cell line, as demonstrated by pHjdThd incorporation assay (Figs. 1 and 2).
No gross gene rearrangements were detected by Southernblot analysis using Bgll-, Bgll- plus Kpnl-, Hindlll-, and EcoRl-digested DNA from these cell lines (not shown). The Southernblot of ///«¿Ill-digested DNA shown in Fig. 4 indicates thatgene amplification occurred only in Colo 205 cells.
Effect of c-myb Sense and Antisense Oligodeoxynucleotideson the Proliferation of Colon Carcinoma Cell Lines. As shownin Fig. 2A, cell number-proliferating LoVo, LoVo/Dx, and Colo205 cells exposed to the highest dose of c-myb AS oligodeoxynucleotides for 3 days was reduced by 55, 56, and 73% ascompared to that in cells grown in medium alone. [3H]dThd
incorporation was reduced by 84% in LoVo, 83% in LoVo/Dx,and 68% in Colo 205 cells after exposure to AS oligodeoxynucleotides (Fig. 2B). No differences in proliferative activity were
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Fig. 1. A, Northern blot analysis of c-myb expression in colon carcinoma celllines. Lanes a, LoVo/Dx; ¿>,DMF-treated LoVo/Dx; c, LoVo; </, Colo 205; e,HT 29;/ normal colonie mucosa. B, ethidium bromide staining of the same filteras control of the amount of RNA present in each lane. Ordinale, kilobases.
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c-myb ANTISENSE OLIGODEOXYNUCLEOTIDES
detected in untreated or myb sense-treated cells. Proliferationof HT 29 cells in which the expression of c-myb mRNA wasnot detected did not appear to be significantly affected by c-
myb AS treatment (Fig. 2).A second group of experiments was designated to determine
whether lowering the AS doses added to the medium wouldresult in differential inhibition of cell proliferation in the variouscell lines and whether the effect correlated with c-myb mRNA
levels. LoVo/Dx and Colo 205 205 cells were not inhibited bythe lowest dose of AS oligodeoxynucleotides which, instead,was still effective on LoVo cells (Fig. 3). Analysis of c-mybmRNA levels was made in oligodeoxynucleotide-treated anduntreated LoVo/Dx cells. From LoVo/Dx total RNA a 234-nucleotide segment corresponding to the 3'-untranslated region
of c-myb mRNA from nucleotides 2254-2487 was generatedby means of reverse transcription and PCR amplification withhuman c-wye-specific primers (15, 20). Southern blot hybridization of the PCR products to a 50-mer c-myb oligomer (15)revealed the presence of c-myb transcripts in untreated or mybsense-treated cells but not in LoVo/Dx cells exposed to c-myb
antisense oligodeoxynucleotides, demonstrating the specific inhibition of gene expression by antisense oligodeoxynucleotides(Fig. 4). In addition 2-microglobulin mRNA levels were constant in all the samples (not shown).
Effect of DMF Treatment on LoVo/Dx Cells. LoVo/Dx cellstreated with DMF lost their normal cubic morphology andbecame spindle shaped, with protruding appendices, and grewin clumps that tightly adhered to the culture dish. Terminaldifferentiation occurred in 15-25 days and was associated withgrowth arrest, increasing production of desmoplakin, and reduced vimentin levels, as demonstrated by indirect immunoflu-orescence (not shown). During the treatment with DMF weanalyzed the expression of c-myb and found that it remainedhigh for 11 days, in association with a strong cell proliferationrate, documented by histone H3 expression (Fig. 5) and the[3H]dThd incorporation (not shown). When differentiating
Lo Vo/Dx cells stopped proliferating, the expression of histoneH3 and c-myb sharply decreased and remained very low untilthe cells became fully differentiated (Figs. 1 and 6). In the sameperiod the mRNA levels of the gene CEA, which is known to
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Fig. 2. Effect of treatment with AS versus S oligodeoxynucleotides or normal culture medium on colon carcinoma cell growth estimated by cell count (A) and|3H]thymidine incorporation (B). Experiments were done in duplicate and repeated three times.
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Fig. 3. Dose-dependent effect on proliferation of colon carcinoma cell lines treated with AS or S oligodeoxynucleotides, estimated as percentage of (B) and [3H]-dThd inhibition of cell number incorporation (A).
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Fig. 4. Southern blot analysis of ////iÃ/111-digestedDNA from: a, LoVo/Dx; ¿>,DMF-treated LoVo/Dx; c, LoVo; d, Colo 205; e, HT 29, hybridized with a e-myh probe (0.6-kilobase EcoRl fragment). Ordinate, kilobases.
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Fig. 5. Expression of c-myb in LoVo/Dx cells treated with sense (a) or antisense(¿>)oligodeoxynucleotides or untreated (c). estimated by PCR amplification of c-myb cDNA; d. contains amplified c-myb DNA from the same cells as control, bp,base pair.
be expressed in the LoVo cell line (16) and appears to be relatedto colonie cell differentiation (26), increased during LoVo/Dxterminal differentiation (Fig. 6).
DISCUSSION
Several studies have provided evidence that c-myb is preferentially expressed in immature hematopoietic cells and is important in the regulation of cell proliferation as hematopoieticprogenitors differentiate into precursors and terminally differentiated cells (3, 5, 14). However, c-myb expression has beendetected in many other cell populations, including neoplasticand embryonal tissues, suggesting that c-myb function is notrestricted to hematopoietic cells. Studies in primary humancolon carcinomas have suggested in many cases a correlationbetween detectable c-myb mRNA levels and the tumor growthfraction (9). Since c-myb mRNA was also detected in coloncarcinoma cell lines, we carried out the present studies todetermine whether c-myb expression is required for the proliferation of colon carcinoma lines expressing high levels of c-
myb mRNA. Of the four colon carcinoma cell lines analyzedLoVo, LoVo/Dx, and Colo cells had detectable levels of c-mybtranscripts, whereas in HT 29 and in normal colonie mucosa c-myb mRNA levels were not detectable, c-myb mRNA levels
differed among the cell lines, with the highest expression in
Fig. 6. Expression of c-myb (a), historie H3 (b) and CEA (c) during differentiation of LoVo/Dx cells induced by DMF. Absicca, period of treatment (days)with DMF. The ethidium bromide-stained gel photographed under UV light (d)was used to quantitate total RNA loaded and transferred during Northern blot.Ordinate, kilobases.
Colo 205; the doxorubicin-resistant LoVo/Dx showed a muchhigher expression than the parental LoVo cells. In LoVo/Dxcells, the c-myb transcript resolved as a doublet which wasconfirmed in other, different Northern blots. The significanceof these two transcripts remains unclear, although differencesin the cap site have been reported to modify the length of c-myb transcripts in mouse thymocytes, suggesting a possiblemechanism utilized to regulate the oncogene function (27).DNA analysis revealed amplification of c-myb in Colo 205cells, confirming previously reported findings (10), while theother cell lines showed no evidence of gene alterations. Thefunctional significance of c-myb expression in these colon carcinoma cell lines was investigated by exposure to c-myb ASoligodeoxynucleotides previously shown to inhibit hemopoieticcell growth (13-15). This treatment reduced cell proliferationin all cell lines expressing c-myb (Fig. 2/1). ['HjThymidine
incorporation in the same cell lines was even more sharplyinhibited, suggesting that inhibition of myb expression (Fig. 5)prevented DNA synthesis in most colon carcinoma cells (Fig.2B). Moreover, treatment with different doses of AS for thesame time resulted in a different effect on proliferation of theresponsive cells. LoVo cells which express low levels of c-myb
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c-myh ANTISENSE OLIGODEOXYNUCLEOTIDES
were inhibited at the lowest dose of AS oligodeoxynucleotides,while LoVo/Dx and Colo 205 cells were not affected. Proliferation of the HT 29 cell line in which c-myb mRNA is notexpressed at detectable levels was, as expected, not affected byexposure to c-myb AS oligodeoxynucleotides.
Our results suggest that the proliferation of some neoplasticcell lines of epithelial origin is maintained by high levels of c-myb expression. This notion, in turn, suggests that the role ofc-myb is more general than previously thought, since, whenactivated, it might be involved in the control of cell cycleprogression at the G,/S transition in colon epithelial cells inaddition to hematopoietic cells and mitogen-stimulated normalT-lymphocytes (13, 14, 28). Although the mechanisms involvedin cell cycle regulation are not known, it is likely that the DNA-binding and -transactivating properties of the c-myb protein arethe relevant features for c-myb function (29-31). However, c-
myb expression is not an absolute requirement for the proliferation of neoplastic epithelial cell lines as shown by the complete independence of HT 29 cell proliferation from the synthesis of c-myb protein.
Treatment of LoVo/Dx DM F resulted in a reduction of c-myb expression, indicating that c-myb down-regulation duringdifferentiation is a common phenomenon, the only knownexception being the embryonic stem cell line "cce" in which c-
myb mRNA levels increase following differentiation into em-bryoid bodies (11). In our experimental system, c-myb expression is related to the proliferative capacity of the cells; in factas the differentiating agent DMF reduces the cell proliferationrate documented by the decrease of the mRNA levels of the Sphase-specific histone H3 gene (23), there is also a concomitantdecline of c-myb mRNA expression; these changes in geneexpression occur before the cells have acquired a more differentiated phenotype. When fully differentiated, the LoVo/Dxcells show a reduced synthesis of laminin receptor, a highexpression of CEA, which progressively increases during theDMF treatment, and a stable low level expression of the c-myboncogene.
Our data demonstrate that inhibition of c-myb expressiondoes not induce differentiation of the colon carcinoma celltested and are in agreement with the findings reported for theHL-60 leukemia cell line (13). It is possible, however, that theexposure time to the AS oligomer was too short to allowcommitment to differentiation and the initial slopes of thisprocess, considering that DMF requires 15-25 days to inducedifferentiation. Additional experiments will reveal whether alonger exposure of the cells to c-myb AS oligodeoxynucleotidescan induce differentiation or whether, in conjunction withDMF, they can shorten the time necessary for this process.
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1991;51:2897-2901. Cancer Res Cecilia Melani, Licia Rivoltini, Giorgio Parmiani, et al.
mybThat Express c-Oligodeoxynucleotides in Colon Adenocarcinoma Cell Lines
AntisensemybInhibition of Proliferation by c-
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