Biochemical Society Transactions

The terms ‘antioxidant’, ‘oxidative stress’ and ‘oxidative damage’ are widely used but rarely defined. This brief review attempts to define them and to examine the ways in which oxidative stress and oxidative damage can affect cell behaviour both in vivo and in cell culture, using cancer as an example.

Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2α, a key regulatory step in translational initiation. The activation of one or more of the eIF2α kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNAMet, the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNAMet allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2α kinase system, mRNA stability and cellular chaperone levels.

Glyoxalase I is part of the glyoxalase system present in the cytosol of cells. The glyoxalase system catalyses the conversion of reactive, acyclic α-oxoaldehydes into the corresponding α-hydroxyacids. Glyoxalase I catalyses the isomerization of the hemithioacetal, formed spontaneously from α-oxoaldehyde and GSH, to S-2-hydroxyacylglutathione derivatives [RCOCH(OH)-SG→RCH(OH)CO-SG], and in so doing decreases the steady-state concentrations of physiological α-oxoaldehydes and associated glycation reactions. Physiological substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclic α-oxoaldehydes. Human glyoxalase I is a dimeric Zn2+ metalloenzyme of molecular mass 42 kDa. Glyoxalase I from Escherichia coli is a Ni2+ metalloenzyme. The crystal structures of human and E. coli glyoxalase I have been determined to 1.7 and 1.5 Å resolution. The Zn2+ site comprises two structurally equivalent residues from each domain – Gln-33A, Glu-99A, His-126B, Glu-172B and two water molecules. The Ni2+ binding site comprises His-5A, Glu-56A, His-74B, Glu-122B and two water molecules. The catalytic reaction involves base-catalysed shielded-proton transfer from C-1 to C-2 of the hemithioacetal to form an ene-diol intermediate and rapid ketonization to the thioester product. R- and S-enantiomers of the hemithioacetal are bound in the active site, displacing the water molecules in the metal ion primary co-ordination shell. It has been proposed that Glu-172 is the catalytic base for the S-substrate enantiomer and Glu-99 the catalytic base for the R-substrate enantiomer; Glu-172 then reprotonates the ene-diol stereospecifically to form the R-2-hydroxyacylglutathione product. By analogy with the human enzyme, Glu-56 and Glu-122 may be the bases involved in the catalytic mechanism of E. coli glyoxalase I. The suppression of α-oxoaldehyde-mediated glycation by glyoxalase I is particularly important in diabetes and uraemia, where α-oxoaldehyde concentrations are increased. Decreased glyoxalase I activity in situ due to the aging process and oxidative stress results in increased glycation and tissue damage. Inhibition of glyoxalase I pharmacologically with specific inhibitors leads to the accumulation of α-oxoaldehydes to cytotoxic levels; cell-permeable glyoxalase I inhibitors are antitumour and antimalarial agents. Glyoxalase I has a critical role in the prevention of glycation reactions mediated by methylglyoxal, glyoxal and other α-oxoaldehydes in vivo.

Rho, Rac and Cdc42, three members of the Rho family of small GTPases, each control a signal transduction pathway linking membrane receptors to the assembly and disassembly of the actin cytoskeleton and of associated integrin adhesion complexes. Rho regulates stress fibre and focal adhesion assembly, Rac regulates the formation of lamellipodia protrusions and membrane ruffles, and Cdc42 triggers filopodial extensions at the cell periphery. These observations have led to the suggestion that wherever filamentous actin is used to drive a cellular process, Rho GTPases are likely to play an important regulatory role. Rho GTPases have also been reported to control other cellular activities, such as the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) cascades, an NADPH oxidase enzyme complex, the transcription factors NF-κB (nuclear factor κB) and SRF (serum-response factor), and progression through G1 of the cell cycle. Thus Rho, Rac and Cdc42 can regulate the actin cytoskeleton and gene transcription to promote co-ordinated changes in cell behaviour. We have been analysing the biochemical contributions of Rho GTPases in cell movement and have found that Rac controls cell protrusion, while Cdc42 controls cell polarity.

It is now accepted that activation of Class I PI3Ks (phosphoinositide 3-kinases) is one of the most important signal transduction pathways used by cell-surface receptors to control intracellular events. The receptors which access this pathway include those that recognize growth factors, hormones, antigens and inflammatory stimuli, and the cellular events known to be regulated include cell growth, survival, proliferation and movement. We have learnt a great deal about the family of Class I PI3K enzymes themselves and the structural adaptations which allow a variety of cell-surface receptors to regulate their activity. Class I PI3Ks synthesize the phospholipid PtdIns(3,4,5)P3 in the membranes in which they are activated, and it is now accepted that PtdIns(3,4,5)P3 and its dephosphorylation product PtdIns(3,4)P2 are messenger molecules which regulate the localization and function of multiple effectors by binding to their specific PH (pleckstrin homology) domains. The number of direct PtdIns(3,4,5)P3/PtdIns(3,4)P2 effectors which exist, even within a single cell, creates an extremely complex signalling web downstream of PI3K activation. Some key players are beginning to emerge, however, linking PI3K activity to specific cellular responses. These include small GTPases for the Rho and Arf families which regulate the cytoskeletal and membrane rearrangements required for cell movement, and PKB (protein kinase B), which has important regulatory inputs into the regulation of cell-cycle progression and survival. The importance of the PI3K signalling pathway in regulating the balance of decisions in cell growth, proliferation and survival is clear from the prevalence of oncogenes (e.g. PI3Kα) and tumour suppressors [e.g. the PtdIns(3,4,5)P3 3-phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)] found in this pathway. The recent availability of transgenic mouse models with engineered defects in Class I PI3K signalling pathways, and the development of PI3K isoform-selective inhibitors by both academic and pharmaceutical research has highlighted the importance of specific isoforms of PI3K in whole-animal physiology and pathology, e.g. PI3Kα in growth and metabolic regulation, PI3Kβ in thrombosis, and PI3Kδ and PI3Kγ in inflammation and asthma. Thus the Class I PI3K signalling pathway is emerging as an exciting new area for the development of novel therapeutics.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge and a ‘metabolic master switch’. When activated by ATP depletion, it switches off ATP-consuming processes, while switching on catabolic pathways that generate ATP. AMPK exists as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits, each of which occurs as multiple isoforms. Rising AMP and falling ATP, brought about by various types of cellular stress (including exercise in skeletal muscle), stimulate the system in an ultrasensitive manner. Acetyl-CoA carboxylase (ACC) exists in mammals as two isoforms, termed ACC-1 and ACC-2 (also known as ACC-α and ACC-β). AMPK phosphorylates and inactivates both isoforms at the equivalent site. Knockout mice, and other approaches, suggest that the malonyl-CoA produced by ACC-2 is exclusively involved in regulation of fatty acid oxidation, whereas that produced by ACC-1 is utilized in fatty acid synthesis. Activation of AMPK by cellular stress or exercise therefore switches on fatty acid oxidation (via phosphorylation of ACC-2) while switching off fatty acid synthesis (via phosphorylation of ACC-1). The Drosophila melanogaster genome contains single genes encoding homologues of the α, β and γ subunits of AMPK (DmAMPK) and of ACC (DmACC). Studies in a Drosophila embryonal cell line show that DmAMPK is activated by stresses that cause ATP depletion (oligomycin, hypoxia or glucose deprivation) and that this is associated with phosphorylation of the site on DmACC equivalent to the AMPK sites on mammalian ACC-1 and ACC-2. This is abolished when expression of DmAMPK is ablated using an RNA interference approach, proving that DmAMPK is necessary for phosphorylation of DmACC in response to ATP depletion.

Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This ‘two-faced’ character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions.

HSL (hormone-sensitive lipase) is a key enzyme in the mobilization of fatty acids from acylglycerols in adipocytes as well as non-adipocytes. In adipocytes, catecholamines stimulate lipolysis mainly through PKA (protein kinase A)-mediated phosphorylation of HSL and perilipin, a protein coating the lipid droplet. The anti-lipolytic action of insulin is mediated mainly via lowered cAMP levels, accomplished through activation of phosphodiesterase 3B. Phosphorylation of HSL by PKA occurs at three sites, the serines 563, 659 and 660, both in vitro and in primary rat adipocytes. Phosphorylation of Ser-659 and -660 is required for in vitro activation as well as translocation from the cytosol to the lipid droplet, whereas the role of the third PKA site remains elusive. Adipocytes isolated from homozygous HSL-null mice, generated in our laboratory, exhibit completely blunted catecholamine-induced glycerol release and reduced fatty acid release, suggesting the presence of additional, although not necessarily hormone-activatable, triacylglycerol lipase(s). Basal hyperinsulinaemia, release of exaggerated amounts of insulin during glucose challenges and retarded glucose disposal during insulin tolerance tests suggest that HSL-null mice are insulin resistant. Liver, adipose tissue and skeletal muscle appear all to be sites of impaired insulin sensitivity in HSL-null mice.

Degeneration of the intervertebral disc has been implicated in chronic low back pain. Type II collagen and proteoglycan (predominantly aggrecan) content is crucial to proper disc function, particularly in the nucleus pulposus. In degeneration, synthesis of matrix molecules changes, leading to an increase in the synthesis of collagens type I and III and a decreased production of aggrecan. Linked to this is an increased expression of matrix-degrading molecules including MMPs (matrix metalloproteinases) and the aggrecanases, ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) 1, 4, 5, 9 and 15, all of which are produced by native disc cells. Importantly, we have found that there is a net increase in these molecules, over their natural inhibitors [TIMP-1 (tissue inhibitor of metalloproteinases-1), 2 and 3], suggesting a deregulation of the normal homoeostatic mechanism. Growth factors and cytokines [particularly TNFα (tumour necrosis factor α) and IL-1 (interleukin 1)] have been implicated in the regulation of this catabolic process. Our work has shown that in degenerate discs there is an increase in IL-1, but no corresponding increase in the inhibitor IL-1 receptor antagonist. Furthermore, treatment of human disc cells with IL-1 leads to a decrease in matrix gene expression and increased MMP and ADAMTS expression. Inhibition of IL-1 would therefore be an important therapeutic target for preventing/reversing disc degeneration.