Inhibition of c-Myc Oncoprotein Limits the Growth of Human Melanoma Cells by Inducing Cellular Crisis
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m304597200
ISSN1083-351X
AutoresAnnamaria Biroccio, Sarah Amodei, A. Antonelli, Barbara Benassi, Gabriella Zupi,
Tópico(s)Cancer Research and Treatments
ResumoHere, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated β-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with l-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-l-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with l-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress. Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated β-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with l-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-l-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with l-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress. The proliferative potential of normal cells in culture is limited to a finite number of population doublings, a phenomenon known as cellular senescence or Hayflick limit (1Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4330) Google Scholar) that is characterized by a large, flat morphology, telomere shortening, a high frequency of nuclear abnormality and induction of β-galactosidase activity (2Dimri G.P. 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This is consistent with the findings that either activation of oncogenes or loss of tumor suppressor function is necessary to override the repression of telomerase in primary somatic cells. The c-myc oncogene has been implicated in the regulation of telomerase through its ability to induce the transcriptional activation of hTERT (25Wang J. Ying Xie L. Allan S. Beach D. Hannon G.J. Genes Dev. 1998; 12: 1769-1774Crossref PubMed Scopus (575) Google Scholar, 26Wu K.J. Grandori C. Amacker M. Simon-Vermot N. Polack A. Lingner J. Dalla-Favera R. Nat. Genet. 1999; 21: 220-224Crossref PubMed Scopus (775) Google Scholar, 27Greenberg R.A. O'Hagan R.C. Deng H. Xiao Q. Hann S.R. Adams R.R. Lichtsteiner S. Chin L. Morin G.B. DePinho R.A. Oncogene. 1999; 18: 1219-1226Crossref PubMed Scopus (359) Google Scholar). By using melanoma-derived clones expressing low levels of c-Myc, we recently demonstrated that telomerase plays a critical role in the c-Myc-dependent tumorigenicity (28Biroccio A. Amodei S. Benassi B. Scarsella M. Cianciulli A. Mottolese M. Del Bufalo D. Leonetti C. Zupi G. Oncogene. 2002; 21: 3011-3019Crossref PubMed Scopus (28) Google Scholar). We have also demonstrated that the down-regulation of c-Myc decreases the intracellular glutathione (GSH) content, resulting in apoptosis (29Biroccio A. Benassi B. Filomeni G. Amodei S. Marchini S. Chiorino G. Rotilio G. Zupi G. Ciriolo M.R. J. Biol. Chem. 2002; 277: 43763-43770Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In this paper we found that GSH depletion and telomere loss induced by c-Myc down-regulation cause the growth arrest of M14 human melanoma cells. Cell Culture—The stable transfectants expressing low levels of c-Myc protein (MAS51, MAS53, and MAS57) and the control clone (MN2) were previously obtained by transfecting M14 parental line with an expression vector carrying antisense c-myc cDNA and/or a neomicine selection marker gene (30Biroccio A. Benassi B. Amodei S. Gabellini C. Del Bufalo D. Zupi G. Mol. Pharmacol. 2001; 60: 174-182Crossref PubMed Scopus (73) Google Scholar). The M14-derived doxycycline-inducible clones expressing either low c-Myc or the puromycin resistance gene were recently obtained by a double transfection with a commercial inducible TET-ON gene expression system (Clontech) consisting of two expression vectors, one a regulator and the other a response vector carrying c-myc cDNA in antisense orientation or only the selected marker (29Biroccio A. Benassi B. Filomeni G. Amodei S. Marchini S. Chiorino G. Rotilio G. Zupi G. Ciriolo M.R. J. Biol. Chem. 2002; 277: 43763-43770Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Doxycycline (Dox; 1 μg/ml) was administered every 24 h either in the clone carrying the empty vector (control clone) or in the c-Myc transfectant. The M14-derived stable transfectants expressing low levels of telomerase activity (MDN2, MDN4, and MDN16) and the control clone (MV3) were previously obtained by transfecting the dominant negative form of hTERT (DN-hTERT) cDNA (kindly provided by Dr. Weinberg) or the puromycin resistance gene only (28Biroccio A. Amodei S. Benassi B. Scarsella M. Cianciulli A. Mottolese M. Del Bufalo D. Leonetti C. Zupi G. Oncogene. 2002; 21: 3011-3019Crossref PubMed Scopus (28) Google Scholar). Serial cell cultivation was done plaiting 105 cells on 6-cm-diameter dishes; 5 days later the total number of cells in the dish were counted, and 105 cells were replated again. This procedure was repeated for 18 in vitro passages. The increase in population doubling level (ΔPDL) was calculated according to the formula (ΔPDL = log(nf /n 0)/log2, where n 0 is the initial number of cells, and nf is the final number of cells. 5mm N-acetyl-l-cysteine (NAC; Sigma) or 1 mm l-buthionine sulfoximine (BSO; Sigma), doses with no toxic effect on cell survival, were used. According to the different experiments, NAC or BSO was added to the culture 24 h after plating and left in the medium for 24 h. The cells were then washed and incubated with fresh medium for 4 days. This procedure was repeated every culture passage. Intracellular GSH content was measured by a colorimetric assay (Bioxytech GSH-400; Oxis International, Inc., Portland, OR), as described by the manufacturer. Morphological Analysis—Senescence-associated β-galactosidase (SA-β-gal) staining was performed as described by Dimri et al. (2Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5788) Google Scholar). Briefly, the cells were fixed with 2% glutaraldehyde in phosphatebuffered saline for 5 min at room temperature, washed in phosphatebuffered saline, and incubated for several hours in staining solution: 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal), 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, 2 mm MgCl2 in phosphate-buffered saline, pH 6.0. The nuclei were stained with 1 μg/ml Hoechst 22358, and cells were analyzed using a fluorescence microscope. Detection of apoptosis was performed in cytospin preparation by terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay using the ApopDETEK in situ apoptosis detection kit (Enzo Diagnostic, New York, NY) as previously reported (28Biroccio A. Amodei S. Benassi B. Scarsella M. Cianciulli A. Mottolese M. Del Bufalo D. Leonetti C. Zupi G. Oncogene. 2002; 21: 3011-3019Crossref PubMed Scopus (28) Google Scholar). The percentage of apoptotic cells was determined by microscopic examination of TUNEL-treated slide at ×200. For each slide, five fields were examined, and 100 cells in each field were counted. Flow Cytometric Analysis—Cell percentages in the different phases of cell cycle were measured by flow cytometric analysis of propidium iodide-stained nuclei as previously described (31Citro G. D'Agnano I. Leonetti C. Perini R. Bucci B. Zon G. Calabretta B. Zupi G. Cancer Res. 1998; 58: 283-289PubMed Google Scholar) using CELLQuest software (Becton Dickinson, San Jose, CA). The percentage of cells synthesizing DNA was measured by flow cytometry (Becton Dickinson) using bromodeoxyuridin (BrdU; Becton Dickinson) incorporation, as previously described (30Biroccio A. Benassi B. Amodei S. Gabellini C. Del Bufalo D. Zupi G. Mol. Pharmacol. 2001; 60: 174-182Crossref PubMed Scopus (73) Google Scholar). Briefly, the cells were labeled with BrdU at a final concentration of 10 μm for 24 h, and the analysis was carried out at the end of BrdU labeling. The cells were then incubated with 2 μg/ml of mouse anti-BrdU (clone BMC 9318; Roche Applied Science) for 30 min at room temperature, and the BrdU-positive cells were revealed with fluorescein isothiocyanate-conjugated anti-mouse monoclonal antibody (1:20; Dako, SA, Glostrup, Denmark). For ROS content, adherent cells were first assayed for viability and then incubated with 4 μm dihydroethidium (Molecular Probes, Eugene, OR) for 45 min at 37 °C. After incubation, the cells were analyzed by flow cytometry. Western Blot—Western blot and detection were performed as previously reported (30Biroccio A. Benassi B. Amodei S. Gabellini C. Del Bufalo D. Zupi G. Mol. Pharmacol. 2001; 60: 174-182Crossref PubMed Scopus (73) Google Scholar). Briefly, 40 μg of total proteins were loaded on denaturing SDS-PAGE. Immunodetection of c-Myc and hTERT proteins was performed by using mouse anti-c-myc monoclonal antibody (clone 9E10; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000 dilution) and rabbit anti-hTERT polyclonal antibody (clone L-20; Santa Cruz Biotechnology; 1:100 dilution). To check the amount of proteins transferred to nitrocellulose membrane, β-actin was used as control. The relative amounts of the transferred proteins were quantified by scanning the autoradiographic films with a gel densitometer scanner (Bio-Rad) and normalized to the related β-actin amounts. Telomeric Repeat Amplification Protocol (TRAP)—Telomerase enzyme activity was measured with the PCR-based TRAP kit (Intergen Company, Oxford, UK) according to the manufacturer's instructions. To define the sensitivity of the method and the semi-quantitative relationship between protein concentration and ladder band intensity, different amounts of protein extract (from 0.01 to 2 μg) were used for each cell line and for all assays. In all cases, the reaction products were amplified in the presence of a 36-bp internal TRAP assay standard, and each extract was tested for heat sensitivity. Each set of TRAP assay included a control reaction without extract. Southern Blot—Total DNA was isolated using the standard procedure. For each sample, 15 μg of DNA were digested with 40 units of Hinf1 and electrophoresed on 0.8% agarose gel. DNA was denatured, neutralized, transferred to a nylon membrane (Hybond N; Amersham Biosciences), and cross-linked with ultraviolet light. The membrane was hybridized with 5′-end [γ-32P]deoxyadenosine triphosphate-labeled telomeric oligonucleotide probe (TTAGGG)3 at 42 °C for 2 h in a rapid hybridization buffer (QuikHyb hybridization solution; Stratagene, La Jolla, CA). After washing, the filters were autoradiographed (Hyperfilm-MP; Amersham Biosciences) with an intensifying screen at –80 °C for 24 h. The autoradiographs were scanned, and the mean telomere length was calculated as reported by Harley et al. (32Harley C.B. Fucher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4665) Google Scholar). Cytogenetic Analysis—To obtain chromosome preparation, the cells were incubated with 0.1 μg/ml colcemid for 1 h and trypsinized, then incubated with hypotonic 0.075 m KCl for 20 min, fixed with methanol to acetic acid (3:1 v/v), dropped onto frosted microscope slides, and air-dried overnight. End-to-end fusions were evaluated in at least 50 Giemsa-stained metaphases from two simultaneously grown cultures for each line and each treatment. For all the experiments, metaphase preparations of the different cells were performed simultaneously under the same conditions. The χ2 test was used for statistical analysis. c-Myc Down-regulation Causes Growth Arrest of M14 Melanoma Cells—We previously generated stable M14 melanoma transfectants expressing low levels of c-Myc compared with control cells (30Biroccio A. Benassi B. Amodei S. Gabellini C. Del Bufalo D. Zupi G. Mol. Pharmacol. 2001; 60: 174-182Crossref PubMed Scopus (73) Google Scholar). By using these transfectants, we here observed that, starting from culture passage 7, their growth rate slowed down, and then they stopped proliferating. In contrast, the growth kinetic of control cells was unabated (Fig. 1A). This effect was accompanied by a reduction of BrdU incorporation (Fig. 1B); although control cells maintained a high and stable labeling with BrdU (approximately 100%), BrdU-positive cells decreased from approximately 90% to less than 10% in the c-Myc transfectants with the increasing of culture passages. To demonstrate the specific role of c-Myc in this phenomenon, a M14-derived Dox-inducible c-Myc low expressing clone, recently generated (29Biroccio A. Benassi B. Filomeni G. Amodei S. Marchini S. Chiorino G. Rotilio G. Zupi G. Ciriolo M.R. J. Biol. Chem. 2002; 277: 43763-43770Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), was used. As previously reported, after 72 h of Dox administration, the cells showed a reduction of c-Myc protein level by approximately 60% compared with uninduced cells. Decreased c-Myc protein expression following Dox administration caused a growth arrest during the in vitro passages (Fig. 2). On the contrary, control cells showed no change in growth. When Dox was removed in the Dox-induced cells, they exhibited a growth rate similar to that of uninduced cells or the control clone, from the eighth passage onwards. Consistent with this result, c-Myc expression (Fig. 2, inset) was lower in the Dox-induced cells than in the uninduced or control clone, and it increased following Dox withdrawal. Dox-induced cells showed distinctive cell characteristics associated with crisis. As cells passaged, a progressively increased expression of SA-β-gal activity was observed in Dox-induced cells, whereas a negligible amount was found in uninduced cells (Fig. 3A). Moreover, although no alteration in phenotypic characteristics was observed in uninduced and Dox-induced cells at early passages, induced cells became enlarged, often contained multiple nuclei, and had a vacuolated cytoplasm from the eighth passage onwards (Fig. 3B, panel a). Cell cycle analysis revealed elevated forward and side scatters, which reflect increased cell size and granularity (Fig. 3B, panel b), and a cell cycle profile substantially altered (Fig. 3B, panel c). A significant decrease in the percentage of cells in G1 phase was observed, which was the major factor contributing to a reduced ratio of G1 to G2/M cells. Staining of nuclei with Hoechst showed that cells contained multiple or multi-lobulated nuclei (Fig. 3B, panel d). These morphological characteristics predominated until day 5 of culture, and then apoptosis appeared (Fig. 3B, panel e). TUNEL staining detected an increase in the percentage of apoptotic cells with the increasing of culture passages, TUNEL-positive cells reaching 60% at the tenth in vitro passage. c-Myc-induced Crisis Is Associated with an Impairment of Telomerase Function and Alteration of Intracellular Redox State—hTERT expression, telomerase activity, and telomere length were analyzed as functions of long term passage in culture. Western blot analysis (Fig. 4A), a semi-quantitative TRAP assay (Fig. 4B), and terminal restriction fragment (TRF; Fig. 4C) were performed in uninduced and Dox-induced c-Myc transfectant after four, eight, and ten in vitro passages. It is evident that hTERT protein expression and telomerase activity were repressed in the Dox-induced cells compared with control cells. The decreased hTERT expression and telomerase activity were evident at the fourth passage, and they were unabated as cells passaged. Consistent with a reduction of telomerase activity, progressive telomere shortening occurred. As shown in Fig. 4C, TRFs length ranged from approximately 12 to 6 kb, with a mean length of approximately 8 kb in control cells. In contrast, in Dox-induced cells TRFs were significantly shorter already after 4 in vitro passages, ranging from 9 to 3 kb with a mean length of approximately 6 kb, and it reached a mean length of approximately 4 kb at the tenth passage. c-Myc-induced crisis was also associated with alteration in the intracellular redox state. As reported in Fig. 4D, the value of GSH in Dox-induced cells was significantly lower (p < 0.01) than control cells. As a consequence, a significant increase in ROS production was observed (Fig. 4E). The measure of reactive oxygen species revealed that although no change in ROS content was evident in uninduced cells, approximately 30% of ROS was already found after four in vitro passages, and this percentage remained unchanged as cells passaged. Restoration of Intracellular Glutathione Content Allows c-Myc Low Expressing Cells to Escape from Crisis—To determine whether the alteration of redox state influenced the c-Myc-dependent crisis, intracellular GSH content was normalized between control and c-Myc low expressing cells. In particular, control cells were treated with BSO, a specific inhibitor of GSH synthesis, to decrease the GSH levels, and in the opposite experiments, GSH levels were increased in the Dox-induced c-Myc cells by adding NAC, which is known to provide cysteine precursor for GSH synthesis. As shown in the Fig. 5A, the intracellular GSH content was significantly reduced in BSO-treated cells to levels comparable with c-Myc low expressing cells. Nevertheless, BSO-treated cells continued to proliferate, and the growth kinetic showed no change compared with untreated cells (Fig. 5B). NAC treatment of the c-Myc low expressing cells raised their GSH levels to those found in the controls (Fig. 5A), enabling the treated cells to continue proliferating, in contrast to the untreated cells (Fig. 5B). Moreover, NAC-treated cells escaped from crisis at late culture passages (Fig. 5C). In fact, the simultaneous analysis of SA-β-gal-positive and polinucleated cells revealed no significant difference between untreated and NAC-treated c-Myc transfectant at passage 8, the percentage of SA-β-gal-positive and polinucleated cells being approximately 40 and 10%, respectively, in all cells, regardless NAC treatment. On the contrary, as cells passaged, the difference between untreated and NAC-treated cells in terms of SA-β-gal-positive and polinucleated percentages became evident. In fact, whereas approximately 60% of SA-β-gal-positive cells were evident in the untreated cells at passage 10, a reduction by approximately 70% was revealed in the NAC-treated cells, and only a minimal presence of SA-β-gal activity existed at passage 18. Similarly, NAC treatment was able to significantly decrease the percentage of cells with supernumerary nuclei by approximately 75%, the percentage of polinucleated cells being approximately 25 and 5% in untreated and NAC-treated cells, respectively. In addition, the percentage of SA-β-gal-positive and polinucleated cells in NAC-treated cells at passage 18 was similar to that observed in control cells. Glutathione Depletion Cooperates with Telomerase Dysfunction in Inducing Crisis—To test the hypothesis that oxidative stress could cooperate with telomerase dysfunction in the c-Myc-dependent crisis, we directly inhibited telomerase function and GSH levels. To this aim, an experimental model previously generated (28Biroccio A. Amodei S. Benassi B. Scarsella M. Cianciulli A. Mottolese M. Del Bufalo D. Leonetti C. Zupi G. Oncogene. 2002; 21: 3011-3019Crossref PubMed Scopus (28) Google Scholar), and consisting of three clones expressing a catalytically inactive DN-hTERT and a control clone expressing the puromycin gene only, was used. We first evaluated the effect of telomerase inhibition on growth properties (Fig. 6A). The growth kinetic of cells carrying a control retrovirus vector did not differ from those of parental cells (Fig. 1). In contrast, following DN-hTERT introduction, the cells displayed many characteristics of crisis, similar to what had been observed after c-Myc down-regulation (Figs. 1, 2, 3). In fact, as shown in Fig. 6B, the percentage of SA-β-gal cells in DN-hTERT cells progressively increased with the culture passages, reaching a value of approximately 70% at passage 14, whereas it remained lower than 5% in control cells. As expected, DN-hTERT cells showed a reduction of telomerase activity by approximately seven times lower than control cells and telomere shortening (Fig. 6, C and D). On the contrary, no change in either c-Myc protein or intracellular GSH content was observed between control and DN-hTERT cells (Fig. 6, E and F). To demonstrate a cooperative effect between oxidative stress and telomerase dysfunction, the intracellular GSH content was decreased in the DN-hTERT cells, and the growth kinetics of untreated and BSO-treated cells were monitored. BSO treatment in DN-hTERT cells reduced the intracellular GSH content (Fig. 7A) to a level comparable with c-Myc transfectant (Fig. 5A) and accelerated the growth arrest induced by the inhibition of hTERT function (Fig. 7B). In fact, although DN-hTERT cells stopped proliferating after 16 culture passages, the BSO-treated cells had undergone growth arrest six passages before. These data were even more apparent from the analysis of SA-β-gal-positive and polinucleated cells evalua
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