(CANCER RESEARCH 51, 2897-2901, June 1, 1991] Inhibition of Proliferation by c-myb Antisense Oligodeoxynucleotides in Colon Adenocarcinoma 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 Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania [B. C.] ABSTRACT Steady-state mRNA levels of the protooncogene c-myb were measured by 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 LoVo and LoVo/Dx cell lines showed that c-myb mRNA levels were much higher in the doxorubicin-resistant derivative than in the parental line, c- myb antisense Oligodeoxynucleotides inhibited cell proliferation only in the cell lines with detectable mRNA c-myb (LoVo, LoVo/DX, and Colo 205). The dose of antisense exerting inhibitory effect was related to the levels of c-myb mRNA expression. Inhibition of c-myb expression in antisense-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 expression would accompany the process of differentiation. During the treatment with dimethylformamide the expression of c-myb decreased in parallel with the reduction of cell growth, while terminal differentiation of these cells was associated with changes in the expression of carcinoembryonic antigen and laminili receptor genes. Our findings demonstrate that the expression of c-myb is important for the proliferation of colon carcinoma cell lines and suggest that the role of this protooncogene is not restricted to cells of hematopoietic origin but 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 regulating cell proliferation and, perhaps, differentiation. In normal he matopoietic cells, c-myb protein levels vary according to the level of differentiation, with very low levels of c-myb protein being detected in terminally differentiated hematopoietic cells (1, 2). In the mouse Friend erythroleukemia cell line, F-MEL, constitutive expression of c-myb prevents terminal differentia tion, while in human myelogenous leukemia cell lines induced to differentiate, c-myb down-regulation is an early event that precedes differentiation (3-5). In nonhematopoietic cells c-myb expression has been de tected in small cell lung cancer (6), teratocarcinoma (7), neuro blastoma cell lines (8), primary colon tumors (9), and colon carcinoma cell lines (10), suggesting that c-myb expression is not strictly tissue specific but might be associated with cell proliferation in tissues of different origin and might play a role in early embryonal development (11). Although the association of 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 in accordance 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 Grant CH-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. Instituto Nazionale Tumori, Milan, Italy [M.P.C.]. cell populations, the exact role of this gene in proliferation and differentiation is not yet well established. A useful tool to analyze the functional relevance of a gene product is to inhibit its expression by exposing target cells to synthetic Oligodeoxynucleotides complementary to the gene transcript (12). This approach has been used to demonstrate the functional relevance of c-myb expression in the hemopoietic system (13-15). Normal hematopoietic colony formation and proliferation of myelogenous leukemia cell lines were both affected by exposure to c-myb AS3 Oligodeoxynucleotides to an extent that depended on the proliferative capacity and level of differentiation of the cell population tested (13-15). To determine whether c-myb expression is also necessary for the proliferation of epithelial neoplastic cells, we first compared levels 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, we exposed neoplastic cells to c-myb AS Oligodeoxynucleotides and found that inhibition of c-myb expression impairs colon carcinoma cell lines proliferation. Analysis of c-myb expression in the AS-treated LoVo/Dx cell line by the reverse transcriptase polymerase chain reaction (PCR) technique revealed that c-myb mRNA levels were down-regulated by AS treatment. The im paired proliferation did not appear to be associated with cell differentiation, as shown by the morphology of AS-treated cells. In LoVo/Dx cells induced to differentiate by treatment with DMF, differentiation was associated with growth arrest, changes in the cell phenotype, and variation in the expression of 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/Dx subline 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 at 65°C,2 mM glutamine, and antibiotics (MA Bioproducts). Exponen tially growing cells were detached by trypsin-EDTA (Flow) and used to isolate nucleic acids. For Oligodeoxynucleotide treatment, 3x10" cells were 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 medium for 3 days at 40, 30, and 20 Mg/ml, respectively. In the dose-response experiments the cells were exposed to three different decreasing doses of AS to obtain concentrations which were 'A, Vio,and '/«of the initial dosage described above; S oligomers were only given at the highest dose. Control cells were cultured in the same conditions without Oli godeoxynucleotides. After 3 days cells were recovered by mechanical detachment, counted, and analyzed for viability by trypan blue dye exclusion. To determine the cell proliferation rate, 3 x 10" S- and AS- treated and untreated cells were washed after treatment with Oligodeox ynucleotides, 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, di methylformamide; PCR, polymerase chain reaction; CEA, carcinoembryonic antigen; dThd, thymidine. 2897 Research. on February 24, 2016. © 1991 American Association for Cancer cancerres.aacrjournals.org Downloaded from