c-MYC apoptotic function is mediated by NRF-1 target genes
Fred Hutchinson Cancer Research Center, Division of Molecular Medicine, Seattle, Washington 98109, USA|, http://www.100md.com
ABSTRACT|, http://www.100md.com
A detailed understanding of the signaling pathways by which c-Myc elicits apoptosis has proven elusive. In the current study, we have evaluated whether the activation of the mitochondrial apoptotic signaling pathway is linked to c-Myc induction of a subset of genes involved in mitochondrial biogenesis. Cytochrome c and other nuclear-encoded mitochondrial genes are regulated by the transcription factor nuclear respiratory factor-1 (NRF-1). The consensus binding sequence (T/C)GCGCA(C/T)GCGC(A/G) of NRF-1 includes a noncanonical CA(C/T)GCG Myc:MAX binding site. In this study, we establish a link between the induction of NRF-1 target genes and sensitization to apoptosis on serum depletion. We demonstrate, by using Northern analysis, transactivation assays, and in vitro and in vivo promoter binding assays that cytochrome c is a direct target of c-Myc. Like c-Myc, NRF-1 overexpression sensitizes cells to apoptosis on serum depletion. We also demonstrate that selective interference with c-Myc induction of NRF-1 target genes by using a dominant-negative NRF-1 prevented c-Myc-induced apoptosis, without affecting c-Myc-dependent proliferation. These results suggest that c-myc expression leads to mitochondrial dysfunction and apoptosis by deregulating genes involved in mitochondrial function.
[Key Words: Apoptosis; c-MYC; NRF-1; mitochondrial biogenesis; Supplemental material is available at .]5i}}*r, 百拇医药
Introduction5i}}*r, 百拇医药
The c-Myc oncogene, which is dysregulated in many cancers (for review, see Nesbit et al. 1999), functions as a positive regulator of cell proliferation and growth (for review, see Dang 1999; Grandori et al. 2000; Nasi et al. 2001). One of the paradoxes of c-myc overexpression is the resultant sensitization of cells to apoptosis under conditions of serum deprivation or after treatment with DNA-damaging agents. (Evan et al. 1992). Understanding the molecular mechanism underlying c-Myc-induced apoptosis may thus have important implications for treatment of Myc-related cancers (for review, see Prendergast 1999). Earlier studies have demonstrated that c-Myc-induced apoptosis involves loss of mitochondrial membrane potential (Fulda et al. 1999), the release of cytochrome c (Juin et al. 2000) and AIF (Daugas et al. 2000), and activation of caspase-3 (Kagaya et al. 1997). These events are associated with mitochondrial dysfunction, and this apoptosis is inhibited by the Bcl-2 oncoprotein, (Bissonnette et al. 1992). Inhibition of c-Myc apoptosis by Bcl-2 may be linked to inhibition of cytochrome c release, and regulation of mitochondrial membrane potential and permeability (Kluck et al. 1997). Although events downstream of the site of action of Bcl-2 are fairly well characterized, the mechanism by which c-myc overexpression disrupts normal mitochondrial function, leading to the release of cytochrome c, remains unexplained.
Production of functional mitochondria requires proteins derived from both nuclear and mitochondrial genomes (for review, see Poyton and McEwen 1996) and is tightly regulated by a series of transcription factors (for review, see Scarpulla 2002. The DNA recognition site for one of these factors includes a core domain CA(T/C)GCG, which is identical to a noncanonical Myc:MAX binding site (Virbasius et al. 1993; Grandori and Eisenman 1997). This factor, called nuclear respiratory factor 1 (NRF-1), is a member of a unique family of evolutionarily conserved transcription factors that play critical roles in eukaryotic development. These include the zebrafish nrf (Becjker et al. 1998) and the Drosophila erect wing gene product (ewg; DeSimone and White 1993), which are both essential genes, as homozygous knockouts are embryonic lethals (DeSimone and White 1993; Becjker et al. 1998). Human NRF-1 has been shown to transactivate the promoters of a number of mitochondrial-related genes (for review, see Scarpulla 2002). NRF-1 binds as a homodimer to the palindromic GC-rich sequence (T/C)GCGCA(C/T)GCGC(A/G), stable high-affinity binding requires a tandem direct repeat of the T/CGCGCA motif, and maximal binding activity requires phosphorylation (Virbasius et al. 1993; Gugneja and Scarpulla 1997).
One of the most well-studied NRF-1 target genes is cytochrome c, a gene shown to play a critical role in both electron transport and apoptosis. Cytochrome c-null mice are not viable beyond day 10, owing to respiratory insufficiency, and cells derived from these embryos are resistant to apoptosis after a variety of stimuli (Li et al. 2000). The induction of cytochrome c on the addition of serum is associated with enhanced respiration (Herzig et al. 2000). Similarly, other genes are coregulated in response to changes in growth state (for review, see Nelson et al. 1995), leading to changes in the proliferation or differentiation of mitochondria. NRF-1, in combination with other transcription factors, controls the coordinated expression of genes essential for mitochondrial function. This ensures that the cell maintains the correct stoichiometry of respiratory complex subunits and a means for their import and correct assembly in the mitochondria (for review, see Poyton and McEwen 1996). A disruption of this critical balance has a detrimental effect, and examples exist of mitochondrial dysfunctions that result from both reduced (for review, see Schapira and Cock 1999) and increased expression of genes involved in mitochondrial function, such as adenine nucleotide translocase-1 (Bauer et al. 1999) and the mitochondrial hinge protein (Okazaki et al. 1998).
The possibility that c-Myc could stimulate NRF-1 target genes, as a result of similarity in binding sites, has prompted the current work. In this study, we sought to determine if the mitochondrial events that underlie c-Myc-induced apoptosis are initiated by the inappropriate expression of NRF-1 target genes. Here we demonstrate that c-Myc is capable of selectively up-regulating NRF-1 target genes. We also show that under conditions shown to trigger c-Myc-induced apoptosis, overexpression of NRF-1 also sensitizes cells to apoptosis. Finally, we demonstrate that a dominant-negative NRF-1 selectively inhibits apoptosis induced by c-Myc while maintaining the proliferative effects of c-Myc. These results are discussed in the context of broader questions regarding the biology of cell death and the role played by c-Myc in cellular metabolism.2rt%:), http://www.100md.com
Results2rt%:), http://www.100md.com
Induction of NRF-1 target genes in Myc-ERTM cell lines2rt%:), http://www.100md.com
In the current study, we have used the conditional Myc-ERTM protein (Littlewood et al. 1995) in an NIH3T3 cell line to evaluate the ability of c-Myc to induce NRF-1 target genes. This protein is a chimeric fusion protein of full-length c-Myc and a mutant estrogen receptor (ERTM). This receptor has a deletion allowing activation by 4-hydroxytamoxifen (4-OHT) but not by endogenous estrogens. We evaluated the induction of NRF-1 target genes over an 8-h time course in the presence of cycloheximide and 4-OHT, as previous studies have demonstrated that maximal transactivation by c-Myc occurs within this timeframe (Wu et al. 1999). Prior to induction, cells were serum depleted for 48 h. We chose to look at two NRF-1-regulated genes, cytochrome c and mtTFA, as potential c-Myc targets. The NRF-1 binding site in the cytochrome c promoter contains a noncanonical Myc:MAX binding sequence (CATGCG), whereas no known c-Myc binding site is present in the mtTFA promoter. We also tested the induction of NRF-1 to determine if c-Myc influenced the expression of this transcription factor. These experiments were done in the presence of cycloheximide to allow an analysis of direct targets of c-Myc. Cytochrome c levels in the Myc-ERTM line showed a significant induction at 8 h after 4-OHT addition, whereas no induction was seen in the vector control line . In contrast, mtTFA and NRF-1 mRNA levels were not significantly induced on 4-OHT addition , indicating that c-Myc does not transactivate these transcription factors. These results provide further evidence that increases in cytochrome c transcripts are a direct result of c-Myc induction. Results of protein labeling studies, performed under serum-deprived conditions, demonstrate that c-Myc expression also results in increases in cytochrome c protein synthesis. Protein levels of cytochrome c were two- to threefold greater than those of control cells grown under similar conditions .
fig.ommittedm(#q2de, http://www.100md.com
Figure 1. c-Myc transactivation of the NRF-1 target gene cytochrome c. (A) Northern analysis of Myc-ERTM and vector control samples isolated at 0, 2, 4, 6, and 8 h after 4-OHT treatment in the presence of 10 µg/mL cycloheximide. Cells had been serum depleted for 48 h prior to cycloheximide and 4-OHT addition. There are three cytochrome c transcripts of 1.4, 1.1, and 0.7 kb. (B) Graph of the relative level of induction of mRNA over time in Myc-ERTM and vector control lines. Data is corrected for loading by using 18S. This data is representative of three separate experiments. (C) Induction of cytochrome c on serum addition in c-myc- /- and c-myc+/+ cell lines after 48 h of serum depletion. This experiment was repeated twice with similar results. (D) EMSA assay of the ability of Myc:MAX and MAX:MAX to bind to the NRF-1 binding site in the cytochrome c promoter. Myc and MAX were both recombinant forms and used alone at a concentration of 25 ng Myc (lane 2) and 7.5 ng MAX (lane 3) or were combined (lane 4). Samples 5-14 show competition with unlabeled dsDNA samples, including E box (lanes 5,6) a mutated E box (lanes 7,8), wild-type cytochrome c (lanes 9-11), and mutant cytochrome c DNA (lanes 12-14). (E) PCR of input chromatin for c-myc- /- and c-myc+/+ and of chromatin immunoprecipitated with c-Myc antibody, nonimmune serum (mock), and no DNA by using primers for cytochrome c, COX5b, and the negative control albumin. The histograms denote the level of Myc association with cytochrome c and COX5b compared with the albumin control for three replicas. (F) Histograms representing the ability of c-Myc to regulate the cytochrome c promoter through the NRF-1 site or a mutant site in which CATGCG has been mutated to CATTAG. Transfections were done in triplicate, and results are expressed as the fold increase in luciferase activity compared with the empty vector control (pRcCMV) corrected for transfection efficiency, and are representative of four separate experiments. (G) Immunoprecipitation with cytochrome c demonstrates increases in the level of cytochrome c protein synthesis on the activation of c-MYC in serum-deprived cells. Control and MycER cell lysates were immunoprecipitated in the presence of mouse IgG conjugated to agarose or cytochrome c coupled to protein G-Sepharose.(Fionnuala Morrish Christopher Giedt and David Hockenbery)
ABSTRACT|, http://www.100md.com
A detailed understanding of the signaling pathways by which c-Myc elicits apoptosis has proven elusive. In the current study, we have evaluated whether the activation of the mitochondrial apoptotic signaling pathway is linked to c-Myc induction of a subset of genes involved in mitochondrial biogenesis. Cytochrome c and other nuclear-encoded mitochondrial genes are regulated by the transcription factor nuclear respiratory factor-1 (NRF-1). The consensus binding sequence (T/C)GCGCA(C/T)GCGC(A/G) of NRF-1 includes a noncanonical CA(C/T)GCG Myc:MAX binding site. In this study, we establish a link between the induction of NRF-1 target genes and sensitization to apoptosis on serum depletion. We demonstrate, by using Northern analysis, transactivation assays, and in vitro and in vivo promoter binding assays that cytochrome c is a direct target of c-Myc. Like c-Myc, NRF-1 overexpression sensitizes cells to apoptosis on serum depletion. We also demonstrate that selective interference with c-Myc induction of NRF-1 target genes by using a dominant-negative NRF-1 prevented c-Myc-induced apoptosis, without affecting c-Myc-dependent proliferation. These results suggest that c-myc expression leads to mitochondrial dysfunction and apoptosis by deregulating genes involved in mitochondrial function.
[Key Words: Apoptosis; c-MYC; NRF-1; mitochondrial biogenesis; Supplemental material is available at .]5i}}*r, 百拇医药
Introduction5i}}*r, 百拇医药
The c-Myc oncogene, which is dysregulated in many cancers (for review, see Nesbit et al. 1999), functions as a positive regulator of cell proliferation and growth (for review, see Dang 1999; Grandori et al. 2000; Nasi et al. 2001). One of the paradoxes of c-myc overexpression is the resultant sensitization of cells to apoptosis under conditions of serum deprivation or after treatment with DNA-damaging agents. (Evan et al. 1992). Understanding the molecular mechanism underlying c-Myc-induced apoptosis may thus have important implications for treatment of Myc-related cancers (for review, see Prendergast 1999). Earlier studies have demonstrated that c-Myc-induced apoptosis involves loss of mitochondrial membrane potential (Fulda et al. 1999), the release of cytochrome c (Juin et al. 2000) and AIF (Daugas et al. 2000), and activation of caspase-3 (Kagaya et al. 1997). These events are associated with mitochondrial dysfunction, and this apoptosis is inhibited by the Bcl-2 oncoprotein, (Bissonnette et al. 1992). Inhibition of c-Myc apoptosis by Bcl-2 may be linked to inhibition of cytochrome c release, and regulation of mitochondrial membrane potential and permeability (Kluck et al. 1997). Although events downstream of the site of action of Bcl-2 are fairly well characterized, the mechanism by which c-myc overexpression disrupts normal mitochondrial function, leading to the release of cytochrome c, remains unexplained.
Production of functional mitochondria requires proteins derived from both nuclear and mitochondrial genomes (for review, see Poyton and McEwen 1996) and is tightly regulated by a series of transcription factors (for review, see Scarpulla 2002. The DNA recognition site for one of these factors includes a core domain CA(T/C)GCG, which is identical to a noncanonical Myc:MAX binding site (Virbasius et al. 1993; Grandori and Eisenman 1997). This factor, called nuclear respiratory factor 1 (NRF-1), is a member of a unique family of evolutionarily conserved transcription factors that play critical roles in eukaryotic development. These include the zebrafish nrf (Becjker et al. 1998) and the Drosophila erect wing gene product (ewg; DeSimone and White 1993), which are both essential genes, as homozygous knockouts are embryonic lethals (DeSimone and White 1993; Becjker et al. 1998). Human NRF-1 has been shown to transactivate the promoters of a number of mitochondrial-related genes (for review, see Scarpulla 2002). NRF-1 binds as a homodimer to the palindromic GC-rich sequence (T/C)GCGCA(C/T)GCGC(A/G), stable high-affinity binding requires a tandem direct repeat of the T/CGCGCA motif, and maximal binding activity requires phosphorylation (Virbasius et al. 1993; Gugneja and Scarpulla 1997).
One of the most well-studied NRF-1 target genes is cytochrome c, a gene shown to play a critical role in both electron transport and apoptosis. Cytochrome c-null mice are not viable beyond day 10, owing to respiratory insufficiency, and cells derived from these embryos are resistant to apoptosis after a variety of stimuli (Li et al. 2000). The induction of cytochrome c on the addition of serum is associated with enhanced respiration (Herzig et al. 2000). Similarly, other genes are coregulated in response to changes in growth state (for review, see Nelson et al. 1995), leading to changes in the proliferation or differentiation of mitochondria. NRF-1, in combination with other transcription factors, controls the coordinated expression of genes essential for mitochondrial function. This ensures that the cell maintains the correct stoichiometry of respiratory complex subunits and a means for their import and correct assembly in the mitochondria (for review, see Poyton and McEwen 1996). A disruption of this critical balance has a detrimental effect, and examples exist of mitochondrial dysfunctions that result from both reduced (for review, see Schapira and Cock 1999) and increased expression of genes involved in mitochondrial function, such as adenine nucleotide translocase-1 (Bauer et al. 1999) and the mitochondrial hinge protein (Okazaki et al. 1998).
The possibility that c-Myc could stimulate NRF-1 target genes, as a result of similarity in binding sites, has prompted the current work. In this study, we sought to determine if the mitochondrial events that underlie c-Myc-induced apoptosis are initiated by the inappropriate expression of NRF-1 target genes. Here we demonstrate that c-Myc is capable of selectively up-regulating NRF-1 target genes. We also show that under conditions shown to trigger c-Myc-induced apoptosis, overexpression of NRF-1 also sensitizes cells to apoptosis. Finally, we demonstrate that a dominant-negative NRF-1 selectively inhibits apoptosis induced by c-Myc while maintaining the proliferative effects of c-Myc. These results are discussed in the context of broader questions regarding the biology of cell death and the role played by c-Myc in cellular metabolism.2rt%:), http://www.100md.com
Results2rt%:), http://www.100md.com
Induction of NRF-1 target genes in Myc-ERTM cell lines2rt%:), http://www.100md.com
In the current study, we have used the conditional Myc-ERTM protein (Littlewood et al. 1995) in an NIH3T3 cell line to evaluate the ability of c-Myc to induce NRF-1 target genes. This protein is a chimeric fusion protein of full-length c-Myc and a mutant estrogen receptor (ERTM). This receptor has a deletion allowing activation by 4-hydroxytamoxifen (4-OHT) but not by endogenous estrogens. We evaluated the induction of NRF-1 target genes over an 8-h time course in the presence of cycloheximide and 4-OHT, as previous studies have demonstrated that maximal transactivation by c-Myc occurs within this timeframe (Wu et al. 1999). Prior to induction, cells were serum depleted for 48 h. We chose to look at two NRF-1-regulated genes, cytochrome c and mtTFA, as potential c-Myc targets. The NRF-1 binding site in the cytochrome c promoter contains a noncanonical Myc:MAX binding sequence (CATGCG), whereas no known c-Myc binding site is present in the mtTFA promoter. We also tested the induction of NRF-1 to determine if c-Myc influenced the expression of this transcription factor. These experiments were done in the presence of cycloheximide to allow an analysis of direct targets of c-Myc. Cytochrome c levels in the Myc-ERTM line showed a significant induction at 8 h after 4-OHT addition, whereas no induction was seen in the vector control line . In contrast, mtTFA and NRF-1 mRNA levels were not significantly induced on 4-OHT addition , indicating that c-Myc does not transactivate these transcription factors. These results provide further evidence that increases in cytochrome c transcripts are a direct result of c-Myc induction. Results of protein labeling studies, performed under serum-deprived conditions, demonstrate that c-Myc expression also results in increases in cytochrome c protein synthesis. Protein levels of cytochrome c were two- to threefold greater than those of control cells grown under similar conditions .
fig.ommittedm(#q2de, http://www.100md.com
Figure 1. c-Myc transactivation of the NRF-1 target gene cytochrome c. (A) Northern analysis of Myc-ERTM and vector control samples isolated at 0, 2, 4, 6, and 8 h after 4-OHT treatment in the presence of 10 µg/mL cycloheximide. Cells had been serum depleted for 48 h prior to cycloheximide and 4-OHT addition. There are three cytochrome c transcripts of 1.4, 1.1, and 0.7 kb. (B) Graph of the relative level of induction of mRNA over time in Myc-ERTM and vector control lines. Data is corrected for loading by using 18S. This data is representative of three separate experiments. (C) Induction of cytochrome c on serum addition in c-myc- /- and c-myc+/+ cell lines after 48 h of serum depletion. This experiment was repeated twice with similar results. (D) EMSA assay of the ability of Myc:MAX and MAX:MAX to bind to the NRF-1 binding site in the cytochrome c promoter. Myc and MAX were both recombinant forms and used alone at a concentration of 25 ng Myc (lane 2) and 7.5 ng MAX (lane 3) or were combined (lane 4). Samples 5-14 show competition with unlabeled dsDNA samples, including E box (lanes 5,6) a mutated E box (lanes 7,8), wild-type cytochrome c (lanes 9-11), and mutant cytochrome c DNA (lanes 12-14). (E) PCR of input chromatin for c-myc- /- and c-myc+/+ and of chromatin immunoprecipitated with c-Myc antibody, nonimmune serum (mock), and no DNA by using primers for cytochrome c, COX5b, and the negative control albumin. The histograms denote the level of Myc association with cytochrome c and COX5b compared with the albumin control for three replicas. (F) Histograms representing the ability of c-Myc to regulate the cytochrome c promoter through the NRF-1 site or a mutant site in which CATGCG has been mutated to CATTAG. Transfections were done in triplicate, and results are expressed as the fold increase in luciferase activity compared with the empty vector control (pRcCMV) corrected for transfection efficiency, and are representative of four separate experiments. (G) Immunoprecipitation with cytochrome c demonstrates increases in the level of cytochrome c protein synthesis on the activation of c-MYC in serum-deprived cells. Control and MycER cell lysates were immunoprecipitated in the presence of mouse IgG conjugated to agarose or cytochrome c coupled to protein G-Sepharose.(Fionnuala Morrish Christopher Giedt and David Hockenbery)