Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells

“Glucose metabolism has drawn much of the attention (for review see Garber, 2006) from the time when Otto Warburg found that some cancer cells consume more glucose than normal cells and proposed that a failure to make ATP by oxidative phosphorylation is the cause of cancer (Warburg, 1956).”

“S2 visualizes localization of phosphorylated histone H2AX in cells deprived of glutamine or treated with etoposide.”

“For example, citrate is used for the synthesis of fatty acids whose defi ciency might change permeability of mitochondrial membranes and affect their regulation by the Bcl-2 proteins (Green and Kroemer, 2004).”

“Some of the Krebs cycle intermediates, such as fumarate, succinate, and 2-oxoglutarate, can function as messenger and cofactor molecules directly regulating activity of enzymes and thereby contributing to regulation of gene transcription and/or tumorigenesis (Selak et al., 2005; Chen et al., 2006).”

“The differences between cancer and normal cells in consumption and metabolism of basic nutrients have been considered a promising cancer therapy target for several decades (for review see Thompson et al., 2005; Shaw and Cantley, 2006).”

“…review see Baggetto, 1992; Curi et al., 2005), abnormal expression of MYC accelerates cell proliferation and changes metabolism of mitochondria (Morrish et al., 2003; Li et al., 2005), and even a transient decrease in ATP concentration can cause apoptosis (Izyumov et al., 2004; Shaw et al., 2004).”

“The availability of nitrogen sources, and glutamine in particular, regulates the activity of mTOR, a kinase that controls protein synthesis, cell proliferation, and other processes that require nitrogen (for review see Sabatini, 2006).”

“This view was also supported by the observation that depletion of glucose induced apoptosis in rodent cells that were transduced with MYC, an oncogene that is abnormally expressed in many human cancers (Nesbit et al., 1999), but not in the parental cell line (Shim et al.”

“Several hypotheses have been proposed to explain this sensitization (for review see (Adhikary and Eilers, 2005).”

“…review see Baggetto, 1992; Curi et al., 2005), abnormal expression of MYC accelerates cell proliferation and changes metabolism of mitochondria (Morrish et al., 2003; Li et al., 2005), and even a transient decrease in ATP concentration can cause apoptosis (Izyumov et al., 2004; Shaw et al., 2004).”

“To test this possibility we used capillary electrophoresis coupled to mass spectrometry (CE-MS), an approach that estimates concentration of multiple metabolites in one sample (Soga et al., 2003).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al.”

“…glutathione defi ciency Abnormally high activity of MYC could produce suffi cient amounts of reactive oxygen species (ROS) to cause DNA damage, genomic instability, and apoptosis (Vafa et al., 2002), perhaps by affecting expression of mitochondrial proteins (Morrish et al., 2003; Li et al., 2005).”

“Many cancer cells indeed accumulate glucose derivatives faster than normal tissues, the ability that has been used successfully for cancer diagnosis and monitoring (for review see Rohren et al., 2004).”

“This system uses a fusion of MYC with a mutated hormonebinding domain of the estrogen receptor, ERTM-MYC (Eilers et al., 1989; Littlewood et al., 1995).”

“3, A and B), whereas crmA, a viral inhibitor of the extrinsic pathway (Enari et al., 1995), had no effect (Fig.”

“However, the idea that restricting supply of glucose or imitating this restriction can kill cancer cells selectively by depleting ATP is not without its critics (for review see Zu and Guppy, 2004).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al., 2003; Li et al., 2005), thus facilitating apoptosis.”

“…used by the cells in a variety of ways, including oxidation by the Krebs cycle to produce ATP (for review see Baggetto, 1992), providing nitrogen required for nucleotide synthesis, and being a precursor of glutathione, the major nonenzymatic cellular anti-oxidant (for review see Curi et al., 2005).”

“For example, increasing the ratio of Bcl-2 proteins may ease permeabilization of the mitochondria after various kinds of stress (Maclean et al., 2003; Egle et al., 2004).”

“…William Tansey (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). pBABE-hygroER-MYC and pBABE-puro MYC-ER plasmids were described previously (Eilers et al., 1989). cDNAs encoding caspase-9 Cys287 to Ser mutant (caspase 9 dominant negative, C9DN) (Fearnhead et al., 1998), crmA, Bcl-2, and…”

“Some of the Krebs cycle intermediates, such as fumarate, succinate, and 2-oxoglutarate, can function as messenger and cofactor molecules directly regulating activity of enzymes and thereby contributing to regulation of gene transcription and/or tumorigenesis (Selak et al., 2005; Chen et al., 2006).”

“Moreover, only a few percent of the consumed glutamine is used for macromolecular biosynthesis, while the rest is metabolized into molecules, such as lactate, that are released by the cells (for review see Newsholme et al., 1985).”

“The observed diversity was consistent with the fi nding that metabolism of human cells is changed by oncogenes in various ways (Ramanathan et al., 2005) and indicated that how nutrient depletion affects cancers and normal cells may be diffi - cult to conclude by extrapolating fi ndings obtained in…”

“Metabolites were extracted from adherent cells by a procedure adopted from Le Belle et al. (2002).”

“…by acute activation of ectopically expressed MYC vary with cell type We found that depletion of glutamine but not glucose induces MYC-dependent apoptosis in human cells, whereas a previous report indicated that glucose depletion caused MYC-dependent apoptosis in a rat cell line (Shim et al., 1998).”

“Glucose metabolism has drawn much of the attention (for review see Garber, 2006) from the time when Otto Warburg found that some cancer cells consume more glucose than normal cells and proposed that a failure to make ATP by oxidative phosphorylation is the cause of cancer (Warburg, 1956).”

“Therefore, glutamine might support cell viability not by providing biosynthetic precursors, but by enabling the Krebs cycle to function as a catalyst of redox reactions (Kovacevic, 1972; Baggetto, 1992).”

“Retroviruses were produced and used to infect primary cells as described previously (Faleiro and Lazebnik, 2000).”

“by inducing nucleotide defi ciency (Ahluwalia et al., 1990).”

“Cells grown in 12-well plates were collected and washed with cold PBS. ATP was extracted by adding 1 ml of boiling water to the cell pellet as described previously (Yang et al., 2002).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al.”

“MYC is also implicated in regulating concentration of glutathione (Benassi et al., 2006), the most abundant nonenzymatic antioxidant in the cell (Anderson et al.”

“…review see Baggetto, 1992; Curi et al., 2005), abnormal expression of MYC accelerates cell proliferation and changes metabolism of mitochondria (Morrish et al., 2003; Li et al., 2005), and even a transient decrease in ATP concentration can cause apoptosis (Izyumov et al., 2004; Shaw et al., 2004).”

“…A ugust 18, 2017 jcb.rupress.org D ow nloaded from http://doi.org/10.1083/jcb.200703099 Supplemental material can be found at: JCB • VOLUME 178 • NUMBER 1 • 2007 94 and some cancer cell lines require glutamine or its metabolites for survival (for review see Medina, 2001; Fuchs and Bode, 2006).”

“97 the preceding steps of apoptosis (Fearnhead et al., 1998), thus avoiding artifacts associated with cell decomposition.”

“Alternatively, pyruvate, oxaloacetate, and other α-ketoacids could function as antioxidants by binding hydrogen peroxide (O’DonnellTormey et al., 1987; Nath et al., 1995).”

“For example, increasing the ratio of Bcl-2 proteins may ease permeabilization of the mitochondria after various kinds of stress (Maclean et al., 2003; Egle et al., 2004).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al., 2003; Li et al., 2005), thus facilitating apoptosis.”

“This cycle has two main functions: to supply NADH and FADH for oxidative phosphorylation by reducing FAD+ and NAD+ and Figure 8.”

“Some cancer cells can even survive in vitro without glucose at all if supplied with glutamine and nucleosides (Reitzer et al., 1979; Wice et al., 1981; Linker et al., 1985).”

“The more recent fi ndings that glucose metabolism is linked to signaling pathways of cell growth and survival, and apoptosis in particular (for review see Hammerman et al., 2004; Fulda and Debatin, 2007) only reinforced the view of glucose metabolism as the Achilles heel of cancer cells.”

“The second possibility seemed more likely because abnormal expression of MYC sensitizes cells to various agents that induce apoptosis (for review see Pelengaris and Khan, 2003) and because previous reports suggested that depletion of glutamine kills because of stress caused by defi ciency in proteins, ATP, glutathione…”

“The rate of glutamine transport is higher in cancerous than in normal cells (Bode et al., 2002), the activity of glutaminase, the fi rst enzyme in glutamine metabolism, correlates with the growth rates of tumors (Knox et al.”

“…et al., 2002), the activity of glutaminase, the fi rst enzyme in glutamine metabolism, correlates with the growth rates of tumors (Knox et al., 1969; Linder-Horowitz et al., 1969; Perez-Gomez et al., 2005), Defi ciency in glutamine but not glucose induces MYC-dependent apoptosis in human cells”

“Although we found that a defi ciency in oxidative phosphorylation does not cause apoptosis in our system, a change in the mitochondrial NADH/NAD+ and FADH/FAD+ ratios, which we did not measure, could affect other processes, including regulation of gene transcription (for review see Ladurner, 2006) and mitochondrial pe…”

“Because the NADH/NAD ratio determines rates of many processes, from glycolysis to histone modifi cations (for review see Ladurner, 2006), affecting this ratio could induce apoptosis.”

“The more recent fi ndings that glucose metabolism is linked to signaling pathways of cell growth and survival, and apoptosis in particular (for review see Hammerman et al., 2004; Fulda and Debatin, 2007) only reinforced the view of glucose metabolism as the Achilles heel of cancer cells.”

“, 1998), crmA, Bcl-2, and human c-MYC were cloned into a MarX-IV-puro retroviral gene transfer vector (Hannon et al., 1999).”

“html), a defi ciency in the Krebs cycle or its intermediates could have various consequences, including apoptosis, even if the normal ATP concentration is maintained by glycolysis (Albayrak et al., 2003; Zhou et al., 2003).”

“…in our system is either the main source of Krebs cycle intermediates, as in some proliferating normal and cancer cells (Reitzer et al., 1979; Moreadith and Lehninger, 1984; Zielke et al., 1984; Kovacevic et al., 1991; Baggetto, 1992; Petch and Butler, 1994), or regulates the Krebs cycle activity.”

“As expected, depletion of glutamine decreased glutathione concentration in IER-MYC–C9DN cells, as did l-buthionine sulfoximine (BSO), an inhibitor of l-glutamylcysteine synthase (Griffi th and Meister, 1979; Anderson et al., 1997) (Fig.”

“Indeed, our observations indicated that glutamine in our system is either the main source of Krebs cycle intermediates, as in some proliferating normal and cancer cells (Reitzer et al., 1979; Moreadith and Lehninger, 1984; Zielke et al., 1984; Kovacevic et al., 1991; Baggetto, 1992; Petch and Butler, 1994), or regulat…”

“As expected, p53 concentration was induced by DON, 6-methylmercaptopurine riboside (MMPR), an inhibitor of nucleotide synthesis (Stet et al., 1995), etoposide, which causes double-stranded DNA breaks, or by activation of ectopically expressed ERTMMYC (Fig.”

“Apoptosis caused by glutamine depletion is prevented by substrates of the Krebs cycle Because glutamine can be converted via glutamate into 2-oxoglutarate, a Krebs cycle intermediate (Kovacevic, 1972; Baggetto, 1992), depletion of this amino acid could cause a defi ciency of this cycle.”

“4 B), and a report that BSO failed to recapitulate apoptosis caused by glutamine deprivation in a hybridoma (Guerin et al., 2006).”

“The amount of reduced glutathione in cells was measured by fl ow cytometry as described previously (Sen et al., 1999).”

“Impressively, glutamine deprivation from fibroblast cultures essentially eliminates pools of the TCA cycle intermediates fumarate and malate (Yuneva et al., 2007).”

“Likewise, glutamine withdrawal triggers apoptosis of Myc-overexpressing cells (Yuneva et al., 2007).”

“Thus, overexpression of MYC in cancer cells renders them sensitive to glutamine withdrawa”