2-Deoxyglucose: An anticancer and antiviral therapeutic,

but not any more a low glucose mimetic

Hyun Tae Kang, Eun Seong Hwang *

Department of Life Science, University of Seoul, Dongdaemungu, Jeonnongdong 90, Seoul, Republic of Korea 130-743
Received 23 August 2004; accepted 12 July 2005

 

Abstract

2-Deoxyglucose (2-DG), a non-metabolizable glucose analogue, blocks glycolysis and inhibits protein glycosylation. It has been tested in multiple studies for possible application as an anticancer or antiviral therapeutic. The inhibitory effect of 2-DG on ATP generation made it a good candidate molecule as a calorie restriction mimetic as well. Furthermore, 2-DG has been utilized in numerous studies to simulate a condition of glucose starvation. Because 2-DG disrupts glucose metabolism, protein glycosylation, and ER quality control at the same time, a cellular or pathologic outcome could be easily misinterpreted without clear understanding of 2-DG’s effect on each of these aspects. However, the effect of 2- DG on protein glycosylation has rarely been investigated. A recent study suggested that 2-DG causes hyperGlcNAcylation of proteins, while low glucose supply causes hypoGlcNAcylation. In certain aspects of cellular physiology, this difference could be disregarded, but in others, this may possibly cause totally different outcomes.
D 2005 Elsevier Inc. All rights reserved.

Keywords: 2-Deoxyglucose; Glucose metabolism; Calorie restriction; Sp1; O-GlcNAcylation; Transcription
Introduction

2-Deoxy-d-glucose (2-DG) is a synthetic analogue of glucose in which the hydroxyl group at the second position carbon is replaced by a hydrogen. 2-DG inhibits phospho- hexose isomerase (Sols and Crane, 1954; Tower, 1958), the enzyme that converts phosphoglucose to phosphofructose, and thereby, blocks glycolysis at the initiation stage. Therefore, 2- DG is expected to cause depletion of ATP as well as of glucose derivatives required for protein glycosylation. 2-DG also induces the so-called unfolded protein response (UPR) in the endoplasmic reticulum (ER), as does low glucose stress (Welch, 1990; Little et al., 1994).
In numerous studies, interference with glycolysis or con- ditions of nutritional deprivation or energy depletion (feeding cells with low or no glucose) has been simulated in in vitro culture or in animals by adding 2-deoxyglucose in the medium (Munoz-Pinedo et al., 2003; Miller et al., 2002; Healy et al.,
2002; Schwoebel et al., 2002; Merker et al., 2002; Kim et al., 2004 for example). 2-DG, alone or in combination with hypoxia or other tumor therapy tools, effectively blocks growth of tumor cells in animal models (Kern and Norton, 1987; Welch, 1990) and of a variety of human tumor cells (Cay et al., 1992; Mese et al., 2001; Dwarkanath et al., 2001). The antiviral effect of 2-DG has also been demonstrated against a variety of enveloped viruses (Kilbourne, 1959; Courtney et al., 1973). 2- DG also has been tested in animal studies as a possible calorie restriction mimetic (Lane et al., 1998). Despite such an extensive use in a wide variety of situations and physiological applications, the effect of 2-DG has simply been speculated to be based on poor metabolic rate and impaired protein glycosylation. However, the effect on protein glycosylation and related signal pathway has been rarely examined or considered in most of the studies that utilized this molecule. This review provides detailed examples of experimental applications of 2-DG and their shortcomings and introduces recent findings on its effect on protein glycosylation, especially O-GlcNAcylation. This review is also intended to promote

* Corresponding author. Tel.: +82 2 2210 2608; fax: +82 2 2210 2888. E-mail address: [email protected] (E.S. Hwang).
0024-3205/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.07.001
correct understanding on the underlying mechanisms of 2-DG action and provide rationale for further application.
2-Deoxyglucose as a metabolic blocker Application as an anticancer therapeutic
Cancer cells in general have higher rates of glycolysis than normal cells. This property, initially observed by Cori (1925) and Warburg (1930), probably developed through adaptation to limited oxygen supply inside tumor mass due to poor vascularization. This suggests that tumor cells consume more glucose, and therefore, are more vulnerable to low level glucose or its metabolism. (Typically, cell lines established from tumors are cultured in media containing 25 mM glucose, while primary cells are cultured in medium containing 5 mM glucose.) This apparent difference between cancer and normal cells led to the attempts to develop a novel cancer therapeutic strategy, and 2-DG appears to perfectly fit into this scheme. In experimental settings, 2-DG treatment inhibited growth of rat fibrosarcoma (Kern and Norton, 1987), hepatocellular carcinoma (Cay et al., 1992; Geschwind et al., 2004), and other carcinoma cells by itself or with other antitumor protocols (Keenan et al., 2004; Maschek et al., 2004; Maher et al., 2003). This inhibition was accompanied by a decrease in ATP level. For example, when 2-DG at 2 g/kg of body weight was administered i.p. to rats bearing methylcholan- threne-induced sarcomas, the [ATP]/[Pi] ratio in tumor cells dropped by 52% (Karczmar et al., 1992).
As well as glucose depletion, 2-DG has been shown to induce expression of P-glycoprotein (P-gp), encoded by the MDR1 gene, and therefore, may help cancer cells develop chemoresistance (Ledoux et al., 2003). The underlying mechanism is not clearly known, but ER stress response and ROS signaling were suggested to be involved. The development of chemoresistance, together with the likely adverse effects on normal cells due to the block on glycolysis and protein glycosylation (albeit to a limited degree), may create a serious drawback to 2-DG treatment as an anticancer regimen. Unless the treatment effectively eliminates tumors by very early treatment, development of P-gp-hyperactive cells may eliminate the chance to use the conventional chemotherapy.

2-Deoxyglucose as a calorie restriction mimetic

Restriction of calories to two thirds of usual intake extends life span of a variety of short-lived species including yeasts, rotifers, Caenorhabditis elegans, spiders, fish, mice, and rats (Weindruch, 1996; Roth et al., 1999; Merry, 2002). It also delays the onset of age-related diseases such as diabetes, cancer, autoimmune diseases, and help maintain cognitive and locomotive functions of brain in rodents (for review, Lane et al., 2002). Recently, experiments with rhesus monkeys suggest that similar effects are seen in this long-lived primate species (Mattison et al., 2003). So far, CR is the only practical means that allows life span extension and reduction in age-related disease rates. Lane et al. (1998) reasoned that the primary factor responsible for the effects of CR is a reduction in total energy intake, and tested if 2-DG treatment

would elicit the same beneficial effects as CR without the necessity of dieting. In fact, in a more recent study, 2-DG treatment was shown to activate hypothalamic AMPK (AMP- activated protein kinase), a protein which is activated when cellular energy is depleted, and thereby, functions as a cellular energy sensor (Kim et al., 2004). In a six-month study, 2-DG administered at 0.4% of body weight lowered fasting insulin level, body temperature, and weight of Fischer344 rats in a manner analogous to that of CR (Lane et al., 1998). Therefore, the authors claimed that metabolic effects media- ting the CR mechanism can be attained pharmacologically, and that it may indeed be possible to obtain the health- and longevity-promoting effects of the CR without actually decreasing food intake. However, to date there has been no report that 2-DG extends the life span or delays incidence of degenerative diseases so far. Furthermore, the effective dose of 2-DG (0.4% w/w) is not much different from a toxic dose (0.6% w/w) that caused four deaths associated with vacuolar changes in the myocardium. In addition, most of the rats treated with 2-DG at 0.6% experienced severe weight loss and a vacuolar change in the heart. There are multiple possible reasons for these pathological findings such as cell death caused by ATP-depletion or by ER stress response. Other possibilities will be discussed below.

2-DG as an antagonist of protein glycosylation

As an inhibitor of protein glycosylation in ER and an inducer of ER stress response

Studies on cellular or physiological responses to levels of glucose suggest that glucose level affects the expression of several genes. Best known are those of the glucose-regulated proteins (Grps) (Little et al., 1994 for review). Two Grps, Grp78 (also known as BiP) and Grp94 are overexpressed in ER of the cells underfed with glucose. Levels of these proteins increased in acidosis and hypoxic conditions as well, and their suppression resulted in increased cell death (Szegezdi et al., 2003 for review). Therefore, Grps are believed to protect cells under stress conditions. Tunicamycin and glucosamine, well known glycosylation antagonists, also induce Grps (Lee, 2001). All these stresses cause an accumulation of misfolded proteins in ER, which, in turn, leads to an unfolded protein response (UPR; or ER stress response) that includes apoptotic cell death in severe cases. In 2-DG-fed cells, lipid-linked oligosaccharides of decreased molecular weight were also formed, and these oligosacharides were not transferred to protein (Datema and Schwarz, 1979). Therefore, 2-DG is expected to induce accumulation of misfolded proteins in ER, and indeed does induce UPR (Lin et al., 1993). In addition, Grp mRNA abundance was reduced in liver, kidney and muscles of CR mice that were fed 20–40% less glucose (Spindler et al., 1990), suggesting a possible complexity of the Grp responses in vivo and in vitro.
In addition to Grp genes, certain genes in glucose metabolism are involved in specific responses to abnormally low glucose level. For example, expression of the genes for
insulin receptor and IGF-1 receptor are down-regulated in cells under glucose starvation (HepG2, a hepatoma cell line, was fed with 5.5 mM instead of 25 mM glucose)(Briata et al., 1990; Briata and Gherzi, 1990). In these studies, 2-DG was employed to demonstrate that inhibition in protein glycosylation is responsible for the decreased gene expression (Briata et al., 1990). Likewise, in many such studies, 2-DG has been employed as a glycosylation antagonist. However, there are multiple different forms of protein glycosylation including O- linked modification with N-acetylglucosamine (O-GlcNAcyla- tion) which takes place in cytosol or nucleus rather than in ER (see below). Certainly, the cellular pathways associated with O- GlcNAcylation of cytosolic/nuclear proteins are expected to be different from that associated with the UPR.

O-GlcNAcylation, a non-ER type protein glycosylation Among a variety of glycoprotein linkages, three types of
protein glycosylations are most widely distributed and well characterized (reviewed in Spiro, 2002). N-linked glycosyla- tion results from transfer of oligosaccharides to asparagine residues en bloc in ER, and is inhibited by tunicamycin. This modification is the one that has been believed most vulnerable to glucose starvation or glycosylation inhibition, leading to UPR. O-linked glycosylation is transfer of a small number of glucose, galactose, or N-acetylgalactosamine to serine or threonine residues in succession during transport through Golgi network. Another form of O-linked protein glycosylation occurs in a variety of proteins including nuclear pore complex proteins, cytoskeletal proteins, and transcription factors (reviewed in Hart, 1997; Van den Steen et al., 1998). This modification consists of addition of a single monosaccharide,
N-acetylglucosamine (GlcNAc) at serine or threonine residues, and is called O-GlcNAcylation.

Effects of 2-DG on protein O-GlcNAcylation

O-GlcNAcylation is catalyzed by UDP-N-acetylglucosami- ne:peptide N-acetylglucosaminyl transferase (O-GlcNAc trans- ferase; OGT) (Haltiwanger et al., 1992). Approximately 2–3% of glucose in the cell is converted to UDP-GlcNAc, the substrate of this enzyme, via the hexosamine biosynthetic pathway (McClain, 2002). The availability of UDP-GlcNAc correlates with glycosylation levels of many intracellular proteins and transcriptional levels of a number of genes (Boehmelt et al., 2000). Therefore, it has been predicted that O-GlcNAcylation of certain transcription factors could regulate gene expression in response to glucose flux (Jackson and Tjian, 1988).
The effect of 2-DG on protein O-GlcNAcylation at the molecular level was only recently investigated (Kang et al., 2003). 2-DG has been suspected to affect Sp1, a ubiquitous transcription factor. As is the case for many cytosolic or nuclear proteins modified by O-GlcNAcylation, Sp1 is modified at multiple serine/threonine residues by monomers of N-acetyl- glucosamine (Jackson and Tjian, 1988; Bouwman and Phili- psen, 2002). And, indeed, the activity of the Sp1-dependent promoters including that of p21WAF1 and human papilloma- virus (HPV) URR (upstream regulatory region), which are well known to be regulated by Sp1, was inhibited by 45 mM 2-DG as well as by low glucose (5 mM) (Kang et al., 2003) (Fig. 1A). Therefore, 2-DG had been correctly predicted to inhibit Sp1. However, the mechanism of Sp1 activity modulation by 2-DG is different from that of low glucose. Both the level of Sp1 protein and its DNA binding activity decreased in cells fed with low glucose, but not in cells treated with 2-DG. Instead, Sp1 protein was highly O-GlcNAcylated and stabilized in 2-DG treatment (Fig. 1B and C). Therefore, 2-DG causes hyper- GlcNAcylation instead of hypoGlcNAcylation of Sp1. Mean-
(A)
(-) 2-DG low Glc

(C)
(-) 2-DG lowGlc

HPV18 E6
Sp1
beta-actin
(B)
O-GlcNAc
1
2 3

(-) 2 4 8 16 24h

Sp1
O-GlcNAc

Sp1
2-DG
low Glc

 

free
probe

1 2 3 4 5 6 1 2 3

Fig. 1. Differences between the effects exerted by 2-DG and low glucose on Sp1 at molecular level. (A) Effect of 2-DG and low glucose treatment on Sp1-dependent transcription. RNA was isolated from HeLa cells incubated in the presence of 25.5 mM glucose and 45 mM 2-DG (lane 2) or 1 mM glucose (lane 3), and applied to RT-PCR for HPV18 E6 whose transcription is dependent on Sp1. RT-PCR on h-actin mRNA was carried out as a control. (B) Effects on Sp1 O-GlcNAcylation and stability. HeLa cells were treated with 2-DG (upper box) or 1 mM glucose (lower box) for an indicated period of time. Nuclear extracts were made, and immunoprecipitated with anti-Sp1 antibody. The immunoprecipitates were applied to Western blotting analysis for O-GlcNAc level (O-GlcNAc) or Sp1 level (Sp1). (C) Effects on Sp1 DNA binding activity. The nuclear extracts (from 24 h time points) were applied to electrophoretic mobility shift assay (EMSA) with [32P]- radiolabelled oligonucleotide whose sequence is present on HPV18 URR (adopted from Kang et al., 2003).
while, Sp1 activity and its GlcNAcylation status were not altered by tunicamycin, an antagonist for N-linked glycosyla- tion (HT Kang, unpublished result). Therefore, the action mode of 2-DG is certainly different from that of low glucose or other glycosylation antagonists.
It has been reported that GlcNAcylation competes with phosphorylation for the same serine/threonine residues in Sp1 (Kamemura and Hart, 2003). Furthermore, Sp1 transactivation capacity is augmented by phosphorylation probably through cyclinA-CDK or MAPK signaling pathway (Fojas de Borja et al., 2001; Merchant et al., 1999; Noe et al., 2001; De Siervi et al., 2004). Therefore, hyperGlcNAcylation, may inhibit the function of Sp1 as a transcription activator by causing hypophosphorylation (Yang et al., 2001). Differently from the hypoGlcNAcylated Sp1 in glucose-starved cells, hyper- GlcNAcylated Sp1 still maintains DNA binding activity, suggesting that hyperGlcNAcylation may disrupt Sp1 inter- action with critical transactivator proteins. Sp1 is known to interact with TATA-binding protein (TBP) and the TBP- associated factor TAFII130 via the glutamine-rich A and B domains (Miller et al., 2002; Munoz-Pinedo et al., 2003), which contain multiple serine/threonine residues (Kadonaga et al., 1988). Kudlow’s group proposed that O-GlcNAcylation of Sp1 activation domain blocks its homomultimerization or interaction with TAFII130, and thereby, interferes with Sp1- driven transcription (Roos et al., 1998; Yang et al., 2001).
How does 2-DG cause hyperGlcNAcylation of Sp1? It turned out that 2-DG inhibits O-GlcNAc-beta-N-acetylgluco- saminidase (O-GlcNAcase), which removes N-acetylgluco- samine residues, and thereby force the equilibrium in favor of GlcNAcylation (Kang et al., 2003). In 2-DG treated cells, overall protein GlcNAcylation increases suggesting that 2-DG could modulate the GlcNAcylation status of many if not all of the so-modified proteins. How GlcNAse is inhibited by 2-DG is not known yet. Another possibility is that 2-DG is converted to UDP-deoxyglucose and gets attached to the serine/threonine residues in place of N-acetylglucosamine. This has not been tested yet, but the conversion of 2-DG to UDP-deoxyglucose has been observed (Biely and Bauer, 1966).
Among many genes modulated by Sp1 are those encoding the insulin receptor (Araki et al., 1991) and the IGF-1 receptor (Beitner-Johnson et al., 1995). As mentioned above, these genes have been shown to be down-regulated by the treatment of low glucose or 2-DG (Briata et al., 1990; HJ Cho and ES Hwang, unpublished result). This supports the idea that Sp1, through GlcNAcylation, plays a role of molecular sensor that allows cells to adapt to level of glucose supply and adjust rates of glucose metabolism.

Possible implication of Sp1 hyperGlcNAcylation by 2-DG 2-DG as an antiviral therapeutic
The antiviral effect of 2-DG was recognized early. Inhibition of multiplication has been reported for some enveloped viruses such as influenza virus (Kilbourne, 1959), sindbis virus and semliki forest virus (Kaluza et al., 1972), herpes simplex virus

(Courtney et al., 1973), respiratory syncytial virus and measles virus (Hodes et al., 1975). Furthermore, 2-DG eliminated genital herpes from most of the tested patients (Blough and Giuntoli, 1979), and cured or decreased the severity of infection of calves with respiratory syncytial virus and infectious bovine rhinotracheitis virus (Tripathy and Mohanty, 1979; Mohanty et al., 1980, 1981). In these early studies, mostly focused on enveloped viruses, inhibition of viral envelope biosynthesis and virion assembly due to blocked glycosylation of membrane proteins appear to be the major mechanism for virus attenuation. This has been supported by altered gel electrophoresis profiles of membrane proteins (Steiner et al., 1973) as well as denuded appearance of budding particles shown by electron microscopy (Tripathy and Mohanty, 1979). In contrast, no alteration in the level of viral RNA synthesis was detected (Kaluza et al., 1972).
More, rather recent, studies suggested that 2-DG can also suppress viral gene expression or viral replication. Tran- scription of the early genes of human papillomavirus type 18 (HPV18) in HeLa cervical carcinoma cells was shown to be inhibited by 2-DG treatment (Maehama et al., 1998; Kang et al., 2003). Inhibition of activity of certain transcription factors that regulate viral gene expression through alterations in its O- GlcNAcylation is a likely basis for this effect. Sp1 was suggested to be a good candidate for such a transcription factor (Kang et al., 2003). Importantly, many different viral genomes have sites that bind to Sp1 in the regulatory regions of critical early genes. Examples are HSV immediate–early gene promoter (Jones and Tjian, 1985), SV40 early promoter (Dynan and Tjian, 1983; Wildeman, 1988), adenovirus E1b promoter (Parks et al., 1988), HIV LTR (Jones and Tjian, 1985; Jones et al., 1986; Harrich et al., 1989; Parrott et al., 1991), HTLV-1 LTR (Gegonne et al., 1993), P4 promoter of the parvovirus minute virus of mice (Pitluk and Ward, 1991), AAV promoter (Pereira and Muzyczka, 1997), hepatitis B virus core and major surface antigen promoters (Raney et al., 1992; Li and Ou, 2001). Therefore, one can reasonably expect that all these viruses can be affected by the treatment with 2-DG.
Sp1 has also been reported to directly interact with and be modulated by certain viral factors. For example, HIV Tat induces Sp1 phosphorylation by DNA protein kinase, and thereby causes an increased activity (Chun et al., 1998). Other viral regulatory proteins also directly interact with Sp1 and activate specific expression of viral and certain cellular genes; SV40 T Ag (Johnston et al., 1996), BPV (Li et al., 1991) and HPV E2 (Steger et al., 2002), HIV-1 Vpr (Wang et al., 1995), HTLV-1 TAX (Trejo et al., 1997), parvovirus NS-1 (Krady and Ward, 1995), and HSV-1 ICP4 proteins (Gu et al., 1993). Although each viral promoter needs to be examined for its sensitivity to Sp1 modulation by 2-DG, this opens a new possibility to an approach to attenuate or shut down virus replication in infected individuals.
Finally, why these viruses have chosen Sp1 among many host’s transcription factors for their own amplification is of an interest. Probably, Sp1, through GlcNAcylation, may allow viruses to check whether the host cell is in condition that is supportive to its active reproduction.
Anticancer
effect
CR effect

Antiviral
effect

 

poor proteasome activity
apoptosis

gRpt2
Sp1-dependent gene expression

 

energy depletion
Glycolysis
block
hyperGlcNAcylation

2-DG
low glucose
hypoGlcNAcylation

Chemo- resistance

Glycosylation
block
pRpt2
high

P-gp induction

ER stress response

Grp induction apoptosis
proteasome activity
CR effect
Fig. 2. Multiple effects of 2-DG and low glucose exerted in a cell. Both 2-DG (bold line) and low glucose (dotted line) cause a block in glycolysis and glycosylation. 2-DG-mediated glycolysis block would lead to apoptotic death of cancer cells (anticancer effect) or to an effect of calorie restriction (CR effect). Both also cause ER stress response which induces P-gp that may help cancer cells develop chemoresistance. 2-DG also causes hyperGlcNAcylation of cellular proteins while low glucose treatment results in hypoGlcNAcylation. In the case of Sp1, both result in a loss of transcription activity. The antiviral effect of 2-DG is likely attributed to the loss of Sp1 activity. In the case of Rpt2, a component of proteasome, low glucose causes its hypoGlcNAcylation that, in turn, leads to an increase in the proteasome activity and rapid turnover of cellular proteins. Meanwhile, 2-DG treatment would induce its hyperGlcNAcylation that is expected to cause proteasome inactivation. Therefore, 2-DG may exert an effect opposite to that is induced by low glucose in the cases of many cellular proteins.
Effects of 2-DG in anti-aging therapy

2-DG treatment down-regulates the expression of genes encoding insulin receptor and IGF-1 receptor in hepatocytes (Briata et al., 1990; HJ Cho and ES Hwang, unpublished result) most likely through inactivation of Sp1 by its hyperGlcNAcy- lation. Inactivation of insulin/IGF-1 signaling cascade has been shown to extend life span of C. elegans, Drosophila, and mice (Longo and Finch, 2003). In addition, IGF-1 (ti/ti ) mice have a physiological state similar to that of calorie-restricted ones, i.e., low plasma levels of insulin, IGF-1, and glucose, and reduced body size and temperature (Longo and Finch, 2003). Therefore, down-regulation of Ins/IGF-1 signaling by 2-DG may potentially provide a certain level of calorie restriction effect. Importantly, calorie restriction itself causes a marked reduction in fasting insulin level, and therefore, in Ins/IGF-1 signaling although it does not cause down-regulation of the receptors in mice (Spindler et al., 2001).
The effect of calorie restriction may also be attributed in part to modulation of the level of GlcNAcylation in certain groups of proteins. The 26S proteasome is a key player mediating turnover of cytosolic and nuclear proteins (Rechsteiner, 2004 for review). The 19S regulatory subcom- plex contains Rpt2 ATPase that is GlcNAcylated, and, its hyperGlcNAcylation inhibits the proteasome activity (Zhang et al., 2003). This result suggests that cellular nutritional status is linked to proteasome activity through GlcNAcylation. When cell is in an adequate nutrition condition, it maintains high GlcNAcylation, and low proteasome activity. Meanwhile,
when starved, cells would contain hypoGlcNAcylated Rpt2 subunit, and thereby, high proteasome activity. Through this pathway, a cell may be able to maintain more efficient use of its resources. Accumulation of undegraded damaged proteins is an important aging process (Gracy, 1985; Berlett and Stadtman, 1997). In aged cells, proteins may accumulate due to low activity of the proteasome, which is responsible for removal of old, damaged proteins (Goto et al., 2001; Carrard et al., 2002). Importantly, in calorie restricted animals, the proteasome activity is somewhat restored (Scrofano et al., 1998). Although there may be multiple explanations for this effect of CR, one may speculate that reduced level of glucose may lead to decreased GlcNAcylation of the Rpt2 ATPase, and to an increase in the proteasome activity. In fact, in glucose-starved cells, the subunit is hypoGlcNAcylated, but highly phosphorylated with increased proteasome activity (Zhang et al., 2003). However, the effect of 2-DG is expected quite the opposite. If 2-DG again causes hyperGlcNAcylation of the Rpt2 ATPase, the proteasome activity is expected to decrease. Therefore, 2-DG treatment would inhibit protein degradation, and thereby make cells accumulate damaged proteins, and this would definitely reduce the potential efficacy of 2-DG as a calorie restriction mimetic even if the toxicity problem could be avoided (Fig. 2).

Conclusion

In most part, 2-DG, by blocking glycolysis and protein glycosylation and causing mild ATP depletion and UPR,
reproduces conditions of cells underfed with glucose (Fig. 1). It also causes aberrant GlcNAcylation of proteins in cytosol and nucleus. 2-DG may be proven a unique tool to study several important issues of cellular and molecular biology and to develop strategies against certain diseases.
First, 2-DG causes protein hyperGlcNAcylation while low glucose causes hypoGlcNAcylation. In Sp1, both result in reduced transcription activity. However, 2-DG may not induce outcomes identical to those caused by low glucose in the cases for other cytosolic or nuclear proteins. Although this possibility has not been studied yet for any proteins, the case of Rpt2 ATPase, the 26S proteasome subunit, may serve a good example. There are scores of proteins known to be modified by O-GlcNAcylation; chaperone proteins including HSP70 and HSP 90; transcription factors including RNA polymerase II large subunit, AP-1, c-Myc, p53, h-catenin, NF-kB, YY1, Rb, CREB; kinases including CKII, GSK3h, PI3kinase; cytoskeletal proteins including keratins, neurofila- ments, myosin, E-cadherin; nuclear pore proteins; proteins involved in glucose metabolism including glycogen synthase, Glut-1, and OGT; and others (eNOS) (Zachara and Hart, 2004, for review). Such studies will certainly help under- standing the significance and function of GlcNAcyl modifi- cation on these proteins.
Second, 2-DG inhibits Sp1 transcription activity without altering DNA binding. These results suggest that proper GlcNAcylation is important for activation of transcription function of Sp1 possibly by modulating protein–protein interaction. Therefore, more information on the GlcNAcy- lated sites on Sp1 induced by 2-DG treatment and on the roles of each sites in target interaction may be utilized in development of drugs that specifically disrupt the interaction between Sp1 and the viral proteins that is necessary for activation of viral gene transcription. For example, if we understand which GlcNAcylation site disrupts Sp1-target interaction, it may be possible to develop a chemical way specifically block the interaction with the viral factors without altering the interaction between Sp1 and cellular factors.
2-DG has been shown to inhibit the O-GlcNAcase enzyme (Kang et al., 2003). So far, O-(2-acetamido-2-deoxy-d- glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) (Haltiwanger et al., 1998) and streptozotocin (STZ) (Han and Kudlow, 1997) are the two best known inhibitors of O- GlcNAcase. 2-DG and STZ caused identical changes on Sp1 O-GlcNAcylation and its activity (Kang et al., 2003). How- ever, there may be certain differences in the target molecules and mechanisms of action. While STZ, an analogue of N- acetylglucosamine, acts as nitric oxide donor and induces apoptotic death of aˆ cells, and causes DNA methylation (Bennett and Pegg, 1981), 2-DG is reasonably believed not to have these activities. However, since 2-DG exerts multiple effects on key cellular functions as well, it is also difficult to correctly assess certain cellular or in vivo outcome at the molecular level. Still, with better understanding on its action mechanisms, 2-DG may be used more safely both experimen- tally and therapeutically.

Acknowledgements

We thank Dr. Daniel DiMaio at Yale University for reviewing the manuscript. This work was supported by the Internal Research Grant of University of Seoul, 2003.

References

Araki, E., Murakami, T., Shirotani, T., Kanai, F., Shinohara, Y., Shimada, F., Mori, M., Shichiri, M., Ebina, Y., 1991. A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene. Journal of Biological Chemistry 266, 3944–3948.
Beitner-Johnson, D., Werner, H., Roberts, C.T. Jr., LeRoith, D., 1995. Regulation of insulin-like growth factor I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element. Molecular Endocrinology 9, 1147–1156.
Bennett, R.A., Pegg, A.E., 1981. Alkylation of DNA in rat tissues following administration of streptozotocin. Cancer Research 41, 2786–2790.
Berlett, B.S., Stadtman, E.R., 1997. Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry 272, 2013–20316.
Biely, P., Bauer, S., 1966. The formation of uridine diphosphate-2-deoxy-d- glucose in yeast. Biochimica et Biophysica Acta 121, 213–214.
Blough, H.A., Giuntoli, R.L., 1979. Successful treatment of human genital herpes infections with 2-deoxy-d-glucose. Journal of the American Medical Association 241, 2798–2801.
Boehmelt, G., Wakeham, A., Elia, A., Sasaki, T., Plyte, S., Potter, J., Yang, Y., Tsang, E., Ruland, J., Iscove, N.N., Dennis, J.W., Mak, T.W., 2000. Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32- deficient cells. EMBO Journal 19, 5092–5104.
Bouwman, P., Philipsen, S., 2002. Regulation of the activity of Sp1-related transcription factors. Molecular and Cellular Endocrinology 195, 27–38.
Briata, P., Gherzi, R., 1990. Multifactorial control of insulin receptor gene expression in human cell lines. Biochemical and Biophysical Research Communications 170, 1184–1190.
Briata, P., Briata, B., Gherzi, R., 1990. Glucose starvation and glycosylation inhibitors reduce insulin receptor gene expression: characterization and potential mechanism in human cells. Biochemical and Biophysical Research Communications 169, 397–405.
Carrard, G., Bulteau, A.L., Petropoulos, I., Friguet, B., 2002. Impairment of proteasome structure and function in aging. International Journal of Biochemistry and Cell Biology 34, 1461–1474.
Cay, O., Radnell, M., Jeppsson, B., Ahren, B., Bengmark, S., 1992. Inhibitory effect of 2-deoxy-d-glucose on liver tumor growth in rats. Cancer Research 52, 5794–5796.
Chun, R.F., Semmes, O.J., Neuveut, C., Jeang, K.T., 1998. Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat. Journal of Virology 72, 2615–2629.
Cori, C.F., 1925. The carbohydrate metabolism of tumors: II. Changes in the sugar, lactic acid, and CO2-combining power of blood passing through a tumor. Journal of Biological Chemistry 65, 397–405.
Courtney, R.J., Steiner, S.M., Benyesh-Melnick, M., 1973. Effects of 2- deoxy-d-glucose on herpes simplex virus replication. Virology 52, 447–455.
Datema, R., Schwarz, R.T., 1979. Interference with glycosylation of glyco- proteins. Inhibition of formation of lipid-linked oligosaccharides in vivo. Biochemical Journal 184, 113–123.
De Siervi, A., Marinissen, M., Diggs, J., Wang, X.F., Pages, G., Senderowicz, A., 2004. Transcriptional activation of p21(waf1/cip1) by alkylphospho- lipids: role of the mitogen-activated protein kinase pathway in the transactivation of the human p21(waf1/cip1) promoter by Sp1. Cancer Research 64, 743–750.
Dwarkanath, B.S., Zolzer, F., Chandana, S., Bauch, T., Adhikari, J.S., Muller, W.U., Streffer, C., Jain, V., 2001. Heterogeneity in 2-deoxy-d- glucose-induced modifications in energetics and radiation responses of human tumor cell lines. International Journal of Radiation Oncology, Biology, Physics 50, 1051–1061.
Dynan, W.S., Tjian, R., 1983. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35, 79–87.
Fojas de Borja, P., Collins, N.K., Du, P., Azizkhan-Clifford, J., Mudryj, M.,
2001.Cyclin A-CDK phosphorylates Sp1 and enhances Sp1-mediated transcription. EMBO Journal 20, 5737–5747.
Gegonne, A., Bosselut, R., Bailly, R.A., Ghysdael, J., 1993. Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1. EMBO Journal 12, 1169–1178.
Geschwind, J.F., Georgiades, C.S., Ko, Y.H., Pedersen, P.L., 2004. Recently elucidated energy catabolism pathways provide opportunities for novel treatments in hepatocellular carcinoma. Expert Review of Anticancer Therapy 4, 449–457.
Goto, S., Takahashi, R., Kumiyama, A.A., Radak, Z., Hayashi, T., Takenouchi, M., Abe, R., 2001. Implications of protein degradation in aging. Annals of the New York Academy of Sciences 928, 54–64.
Gracy, R., 1985. Impaired Protein Degradation May Account for the Accumulation of FAbnormal_ Proteins in Aged Cells. Alan R. Hiss Inc., New York, pp. 1–18.
Gu, B., Rivera-Gonzalez, R., Smith, C.A., DeLuca, N.A., 1993. Herpes simplex virus infected cell polypeptide 4 preferentially represses Sp1-activated over basal transcription from its own promoter. Proceedings of the National Academy of Sciences of the United States of America 90, 9528–9532.
Haltiwanger, R.S., Blomberg, M.A., Hart, G.W., 1992. Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyl- transferase. Journal of Biological Chemistry 267, 9005–9013.
Haltiwanger, R.S., Grove, K., Philipsberg, G.A., 1998. Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2- acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Journal of Biological Chemistry 273, 3611–3617.
Han, I., Kudlow, J.E., 1997. Reduced O-glycosylation of Sp1 is associated with increased proteasome susceptibility. Molecular Cell Biology 17, 2550–2558.
Harrich, D., Garcia, J., Wu, F., Mitsuyasu, R., Gonazalez, J., Gaynor, R., 1989. Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat. Journal of Virology 63, 2585–2591.
Hart, G.W., 1997. Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins. Annual Review of Biochemistry 66, 315–335.
Healy, D.A., Watson, R.W., Newsholme, P., 2002. Glucose, but not glutamine, protects against spontaneous and anti-Fas antibody-induced apoptosis in human neutrophils. Clinical Science (London) 103, 179–189.
Hodes, D.S., Schnitzer, T.J., Kalica, A.R., Camargo, E., Chanock, R.M., 1975. Inhibition of Respiratory syncytial, parainfluenza 3 and measles viruses by 2-deoxy-d-glucose. Virology 63, 201–208.
Jackson, S.P., Tjian, R., 1988. O-Glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell 55, 125–133.
Johnston, S.D., Yu, X.M., Mertz, J.E., 1996. The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitia- tion complex. Journal of Virology 70, 1191–1202.
Jones, K.A., Tjian, R., 1985. Sp1 binds to promoter sequences and activates herpes simplex virus Fimmediate-early_ gene transcription in vitro. Nature 317, 179–182.
Jones, K.A., Kadonaga, J.T., Luciw, P.A., Tjian, R., 1986. Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. Science 232, 755–759.
Kadonaga, J.T., Courey, A.J., Ladika, J., Tjian, R., 1988. Distinct regions of Sp1 modulate DNA binding and transcriptional activation. Science 242, 1566–1570.
Kaluza, G., Scholtissek, C., Rott, R., 1972. Inhibition of the multiplication of enveloped RNA-viruses by glucosamine and 2-deoxy-d-glucose. Journal of General Virology 14, 251–259.
Kamemura, K., Hart, G.W., 2003. Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: a new paradigm for

metabolic control of signal transduction and transcription. Progress in Nucleic Acid Research and Molecular Biology 73, 107–136.
Kang, H.T., Ju, J.W., Cho, J.W., Hwang, E.S., 2003. Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose. Journal of Biological Chemistry 278, 51223–51231.
Karczmar, G.S., Arbeit, J.M., Toy, B.J., Speder, A., Weiner, M.W., 1992. Selective depletion of tumor ATP by 2-deoxyglucose and insulin, detected by 31P magnetic resonance spectroscopy. Cancer Research 52, 71–76.
Keenan, J., Liang, Y., Clynes, M., 2004. Two-deoxyglucose as an anti- metabolite in human carcinoma cell line RPMI-2650 and drug-resistant variants. Anticancer Research 24, 433–440.
Kern, K.A., Norton, J.A., 1987. Inhibition of established rat fibrosarcoma growth by the glucose antagonist 2-deoxy-d-glucose. Surgery 102, 380–385.
Kilbourne, E.D., 1959. Inhibition of influenza virus multiplication with a glucose antimetabolite (2-deoxy-d-glucose). Nature 183, 271–272.
Kim, M.S., Park, J.Y., Namkoong, C., Jang, P.G., Ryu, J.W., Song, H.S., Yun, J.Y., Namgoong, I.S., Ha, J., Park, I.S., Lee, I.K., Viollet, B., Youn, J.H., Lee, H.K., Lee, K.U., 2004. Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nature Medicine 10, 727–733.
Krady, J.K., Ward, D.C., 1995. Transcriptional activation by the parvoviral nonstructural protein NS-1 is mediated via a direct interaction with Sp1. Molecular Cell Biology 15, 524–533.
Lane, M.A., Ingram, D.K., Roth, G.S., 1998. A strategy for identifying biomarkers of aging: further evaluation of hematology and blood chemistry data from a calorie restriction study in rhesus monkeys. Journal of Anti- Aging Medicine 1, 327–337.
Lane, M.A., Mattison, J., Ingram, D.K., Roth, G.S., 2002. Caloric restriction and aging in primates: relevance to humans and possible CR mimetics. Microscopy Research and Technique 59, 335–338.
Ledoux, S., Yang, R., Friedlander, G., Laouari, D., 2003. Glucose depletion enhances P-glycoprotein expression in hepatoma cells: role of endoplasmic reticulum stress response. Cancer Research 63, 7284–7290.
Lee, A.S., 2001. The glucose-regulated proteins: stress induction and clinical applications. Trends in Biochemical Sciences 26, 504–510.
Li, J., Ou, J.H., 2001. Differential regulation of hepatitis B virus gene expression by the Sp1 transcription factor. Journal of Virology 75, 8400–8406.
Li, R., Knight, J.D., Jackson, S.P., Tjian, R., Botchan, M.R., 1991. Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell 65, 493–505.
Lin, H.Y., Masso-Welch, P., Di, Y.P., Cai, J.W., Shen, J.W., Subjeck, J.R., 1993. The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin. Molecular Biology of the Cell 4, 1109–1119.
Little, E., Ramakrishnan, M., Roy, B., Gazit, G., Lee, A.S., 1994. The glucose- regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Critical Reviews in Eukaryotic Gene Expression 4, 1–18.
Longo, V.D., Finch, C.E., 2003. Evolutionary medicine: from dwarf model systems to healthy centenarians? Science 299, 1342–1346.
Maehama, T., Patzelt, A., Lengert, M., Hutter, K.J., Kanazawa, K., Hausen, H., Rosl, F., 1998. Selective down-regulation of human papillomavirus transcription by 2-deoxyglucose. International Journal of Cancer 76, 639–646.
Maher, J.C., Krishan, A., Lampidis, T.J., 2003. Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-d-glucose in tumor cells treated under hypoxic vs aerobic conditions. Cancer Chemotherapy and Pharmacology 53, 116–122.
Maschek, G., Savaraj, N., Priebe, W., Braunschweiger, P., Hamilton, K., Tidmarsh, G.F., De Young, L.R., 2004. 2-Deoxy-d-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo. Cancer Research 64, 31–34.
Mattison, J.A., Lane, M.A., Roth, G.S., Ingram, D.K., 2003. Calorie restriction in rhesus monkeys. Experimental Gerontology 38, 35–46.
McClain, D.A., 2002. Hexosamines as mediators of nutrient sensing and regulation in diabetes. Journal of Diabetes and its Complications 16, 72–80.
Merchant, J.L., Du, M., Todisco, A., 1999. Sp1 phosphorylation by Erk 2 stimulates DNA binding. Biochemical and Biophysical Research Commu- nications 254, 454–461.
Merker, M.P., Bongard, R.D., Kettenhofen, N.J., Okamoto, Y., Dawson, C.A.,
2002.Intracellular redox status affects transplasma membrane electron transport in pulmonary arterial endothelial cells. American Journal of Physiology. Lung Cellular and Molecular Physiology 282, L36–L43.
Merry, B.J., 2002. Molecular mechanisms linking calorie restriction and longevity. International Journal of Biochemistry and Cell Biology 34, 1340–1354.
Mese, H., Sasaki, A., Nakayama, S., Yokoyama, S., Sawada, S., Ishikawa, T., Matsumura, T., 2001. Analysis of cellular sensitization with cisplatin- induced apoptosis by glucose-starved stress in cisplatin-sensitive and – resistant A431 cell line. Anticancer Research 21, 1029–1033.
Miller, C.C., Martin, R.J., Whitney, M.L., Edwards, G.L., 2002. Intracere- broventricular injection of fructose stimulates feeding in rats. Nutritional Neuroscience 5, 359–362.
Mohanty, S.B., Rockemann, D.D., Tripathy, R.N., 1980. Chemotherapeutic value of 2-deoxy-d-glucose in infectious bovine rhinotracheitis viral infection in calves. American Journal of Veterinary Research 41, 1049–1051.
Mohanty, S.B., Rockemann, D.D., Davidson, J.P., Tripathy, R.N., Ingling, A.L., 1981. Chemotherapeutic effect of 2-deoxy-d-glucose against respiratory syncytial viral infection in calves. American Journal of Veterinary Research 42, 336–338.
Munoz-Pinedo, C., Ruiz-Ruiz, C., Ruiz De Almodovar, C., Palacios, C., Lopez- Rivas, A., 2003. Inhibition of glucose metabolism sensitizes tumor cells to death receptor-triggered apoptosis through enhancement of death-inducing signaling complex formation and apical procaspase-8 processing. Journal of Biological Chemistry 278, 12759–12768.
Noe, V., Alemany, C., Nicolas, M., Ciudad, C.J., 2001. Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resist- ance to methotrexate in Chinese hamster ovary cells. European Journal of Biochemistry 268, 3163–3173.
Parks, C.L., Banerjee, S., Spector, D.J., 1988. Organization of the transcrip- tional control region of the E1b gene of adenovirus type 5. Journal of Virology 62, 54–67.
Parrott, C., Seidner, T., Duh, E., Leonard, J., Theodore, T.S., Buckler-White, A., Martin, M.A., Rabson, A.B., 1991. Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T lymphocytes. Journal of Virology 65, 1412–1414.
Pereira, D.J., Muzyczka, N., 1997. The cellular transcription factor SP1 and anAˆunknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter. Journal of Virology 71, 1747–1756.
Pitluk, Z.W., Ward, D.C., 1991. Unusual Sp1-GC box interaction in a parvovirus promoter. Journal of Virology 65, 6661–6670.
Raney, A.K., Le, H.B., McLachlan, A., 1992. Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 tran- scription factor. Journal of Virology 66, 6912–6921.
Rechsteiner, M.C., 2004. Ubiquitin-mediated proteolysis: an ideal pathway for systems biology analysis. Advances in Experimental Medicine and Biology 547, 49–59.
Roos, M.D., Xie, W., Su, K., Clark, J.A., Yang, X., Chin, E., Paterson, A.J., Kudlow, J.E., 1998. Streptozotocin, an analog of N-acetylglucosamine, blocks the removal of O-GlcNAc from intracellular proteins. Proceedings of the Association of American Physicians 110, 422–432.
Roth, G.S., Ingram, D.K., Lane, M.A., 1999. Calorie restriction in primates: will it work and how will we know? Journal of American Geriatric Society 47, 896–903.
Schwoebel, E.D., Ho, T.H., Moore, M.S., 2002. The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion. Journal of Cell Biology 157, 963–974.

Scrofano, M.M., Shang, F., Nowell, T.R. Jr., Gong, X., Smith, D.E., Kelliher, M., Dunning, J., Mura, C.V., Taylor, A., 1998. Aging, caloric restriction and ubiquitin-dependent proteolysis in the livers of Emory mice. Mechanism of Ageing and Development 101, 277–296.
Sols, A., Crane, R.K., 1954. Substrate specificity of brain hexokinase. Journal of Biological Chemistry 210, 581–595.
Spindler, S.R., Crew, M.D., Mote, P.L., Grizzle, J.M., Walford, R.L., 1990. Dietary energy restriction in mice reduces hepatic expression of glucose- regulated protein 78 (BiP) and 94 mRNA. Journal of Nutrition 120, 1412–1417.
Spindler, S.R., Grizzle, J.M., Walford, R.L., Mote, P.L., 2001. Aging and restriction of dietary calories increases insulin receptor mRNA, and aging increases glucocorticoid receptor mRNA in the liver of female C3B10RF1 mice. Journal of Gerontology 46, B233–B237.
Spiro, R.G., 2002. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43–56.
Steger, G., Schnabel, C., Schmidt, H.-M., 2002. The hinge region of the human papillomavirus type 8 E2 protein activates the human p21(WAF1/
CIP1) promoter via interaction with Sp1. Journal of General Virology 83, 503–510.
Steiner, S., Courtney, R.J., Melnick, J.L., 1973. Incorporation of 2-deoxy-d- glucose into glucoproteins of normal and Simian virus 40-transformed hamster cells. Cancer Research 33, 2402–2407.
Szegezdi, E., Fitzgerald, U., Samali, A., 2003. Caspase-12 and ER-stress- mediated apoptosis: the story so far. Annals of the New York Academy of Sciences 1010, 186–194.
Tower, D.B., 1958. The effects of 2-deoxy-d-glucose on metabolism of slices of cerebral cortex incubated in vitro. Journal of Neurochemistry 3, 185–205.
Trejo, S.R., Fahl, W.E., Ratner, L., 1997. The tax protein of human T-cell leukemia virus type 1 mediates the transactivation of the c-sis/platelet- derived growth factor-B promoter through interactions with the zinc finger transcription factors Sp1 and NGFI-A/Egr-1. Journal of Biological Chemistry 272, 27411–27421.
Tripathy, R.N., Mohanty, S.B., 1979. Effect of 2-deoxy-d-glucose and glucosamine on bovine respiratory syncytial virus. American Journal of Veterinary Research 40, 1288–1293.
Van den Steen, P., Rudd, P.M., Dwek, R.A., Opdenakker, G., 1998. Concepts and principles of O-linked glycosylation. Critical Reviews in Biochemistry and Molecular Biology 33, 151–208.
Wang, L., Mukherjee, S., Fengian, J., Narayan, O., Zhao, L.-J., 1995. Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and trans-activation of viral long terminal repeat. Journal of Biological Chemistry 270, 25564–25569.
Warburg, O., 1930. The Metabolism of Tumors. Arnold Constable, London. Weindruch, R., 1996. Caloric restriction and aging. Scientific American 274,
46–52.
Welch, W.J., 1990. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, NY, pp. 223–278.
Wildeman, A.G., 1988. Regulation of SV40 early gene expression. Biochem- istry and Cell Biology 66, 567–577.
Yang, S., Su, K., Roos, M.D., Chang, Q., Paterson, A.J., Kudlow, J.E., 2001. O-Linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability. Proceedings of the National Academy of Sciences of the United States of America 98, 6611–6616.
Zachara, N.E., Hart, G.W., 2004. O-GlcNAc modification: a nutritional sensor that modulates proteasome function. Trends in Cell Biology 14, 218–221.
Zhang, F., Su, K., Yang, X., Bowe, D.B., Paterson, A.J., Kudlow, J.E., 2003. O- GlcNAc modification is an endogenous inhibitor of the proteasome. Cell 115, 715–725.