INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE II: 737-742, 2003 Effect of apoptogenic stimuli on colon carcinoma cell lines with a different c-myc expression level CHIARAGORRINI 1 , MADDALENA DONZELLI 1 , ALICIA TORRIGLIA 2 , ROSANNA SUPIN0 3 , OLIVIER BRISON 4 , ROSA BERNARDI 1 , CLAUDIA NEGRI 1 , MARCO DENEGRI 1 , MARIE-FRANCE COUNIS 2 , G. NADIA RANZANI 5 and A. IVANA SCOVASSI 1 istituto di Genetica Molecolare CNR, Via Abbiategrasso 207,1-27100 Pavia, Italy; 2 U450 INSERM, •3 15 Rue de lEcole de Médecine, 75005 Paris, France; Istituto Nazionale Tumori, Via Venezian 1, 20133 Milano, Italy; 4 Institut Gustave-Roussy, 94805 Villejuif, France; Dipartimento di Genetica, Università di Pavia, Italy Received July 27, 2002; Accepted September 24, 2002 Abstract. We have recently demonstrated that a high c-myc endogenous amplification level confers an apoptosis-prone phenotype to serum-deprived colon carcinoma SW613-S cells. The aim of this study was to gain new insights into the features of c-myodependent apoptosis, by extending our analysis to different apoptogenic stimuli. The study was carried out on clones, derived from the human colon carcinoma SW613-S cell line, which harbor different levels of endogenous c-myc amplification, and on isogenic cell lines with an enforced c-myc overexpression. Our results indicate that cells with endogenous or transfected exogenous c-myc overexpression (SW613-12A1 and -2GlmycP2Tul cell lines, respectively), activate the apoptotic machinery in response to the treatment with eto- poside, doxorubicin and vitamin D 3 , which induce apoptosis through the death receptor Fas. The low levels of c-myc expression present in SW613-B3 and -B3mycC5, seem to be unable to activate Fas-mediated apoptosis, thus suggesting that only a high c-myc expression can bypass the lack of Fas receptor. Apoptosis induction mediated by DNA damage and long-term culture was independent of c-myc expression. A pathway of apoptosis characterized by the activation of the enzyme L-DNase II, was observed in both 12Al and B3 cell lines. Introduction It is generally assumed that anticancer drugs may exert their cytotoxicity through the induction of apoptotic cell death. In this view, the overexpression of anti-apoptotic genes could render tumors refractory to chemotherapy, and the presence Correspondence to: Dr A. Ivana Scovassi, Istituto di Genetica Molecolare CNR, Via Abbiategrasso 207,1-27100 Pavia, Italy E-mail: scovassi@igm.cnr.it Key words: c-myc, colon carcinoma cells, apoptosis, caspases, L-DNase II of pro-apoptotic factors may be efficient in driving tumor cells to apoptosis (reviewed in refs. 1-4). The c-myc proto-oncogene encodes a transcription factor that promotes proliferation, growth and apoptosis (reviewed in refs. 5-8). Normal expression of the c-myc gene is critical for regulating cell proliferation, whereas its deregulated expression, either by gene amplification or by chromosomal translocation, is a hallmark of neoplasia (5-8). The molecular mechanisms leading to c-Myc-mediated apoptosis or cell proliferation are largely unknown. It could be hypothesized that the dual and opposite role of c-Myc is regulated by extracellular signals promoting its interaction with specific molecular partners; such a regulation has been recently proposed for Ras proteins, which can promote either cell survival or apoptosis by the association to different effectors (9). The pro-apoptotic function of c-myc was demonstrated by its enforced overexpression and further confirmed in cell lines harboring different endogenous levels of the oncogene. In a previous work, we investigated the response to serum deprivation of human colon carcinoma cells harboring different endogenous levels of c-myc. We observed that the tumori- genic SW613-12A1 cell line, which is characterized by a high endogenous c-myc amplification, activated the apoptotic pathway, while SW613-B3 cells, with a low level of c-myc amplification, were unable to promote serum deprivation- induced apoptosis (10). By the parallel analysis of isogenic cell lines with enforced exogenous c-myc overexpression, we determined a crucial role of this oncogene in serum deprivation-induced apoptosis (10). The aim of this study was to investigate, in the same experimental model, the relevance of c-myc in apoptosis by i) drugs which activate Fas-mediated apoptosis, i.e. etoposide, doxorubicin and vitamin D 3 ; ii) agents which induce DNA damage, i.e. bleomycin; iii) growth factor depletion determined by long-term culture conditions. We found that 12A1 cells, harboring a high endogenous c-myc amplification level, are generally apoptosis-prone, while B3 (with a low c-myc expression), are able to activate apoptosis only after DNA damage or when cultured long-term, but not in response to etoposide, doxorubicin and vitamin D 3 . Moreover, we showed that the introduction of multiple copies of a plasmid bearing