Under normal homeostatic conditions, Nrf2 is retained in the cytoplasm through its interaction with Keap1. Upon oxidative stress, Nrf2 is released from Keap1, and translocates from the cytoplasm to the nucleus [18]. This release mechanism has been extensively studied and several mechanisms have been proposed including modification of Cys residues in Keap1, phosphorylation at Ser40 by protein kinase C (PKC), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and phosphoinositide-3-kinase (PI3K) [21,22];

Under unstressed conditions, Nrf2 is constantly degraded via Keap1-mediated ubiquitination that is counter balanced by constitutive Nrf2 translocation. Specifically, in addition to binding Nrf2 through its Kelch-repeat domain, Keap1 also binds Cullin-3 (Cul3) [23,24]. In turn, Cul3 binds its partner Rbx1 to form a core E3 ubiquitin ligase complex. This complex targets Nrf2 for degradation. Under oxidative conditions, Keap1-mediated ubiquitination is impeded, while Nrf2 nuclear translocation levels are elevated, thus resulting in binding to the ARE in combination with other mediators of transcription.

After migration to the nucleus, Nrf2 undergoes heterodimeric combinations with other transcription factors, such as small Maf protein and binds to the 5′-upstream cis-acting regulatory sequence, termed ARE or electrophile response elements. However, the role of Nrf2-Maf heterodimers in ARE-mediated gene regulation is controversial and requires further investigation [2,25]. Over the past decade evidence has been accumulating that suggests another control mechanism of Nrf2 activation exists. This is in the form of another transcription factor Bach1 which is known to bind to the Nrf2 site as a heterodimer with Maf [20,26]. Upon induction, Bach1 is replaced by Nrf2, resulting in activation and suggesting competition between Bach1 and Nrf2 for the same DNA binding site in different cellular states [27,28].

This section shows the many studies which have come together to dissect and understand the mechanism involved in regulating Nrf2 activation. In the next section we will inform the reader about the genes that are regulated by Nrf2 activation together with what this means for the cellular environment.

HO-1 is arguably the most well known of all Nrf2-regulated genes. It catalyzes the rate-limiting step in the catabolism of the pro-oxidant heme to carbon monoxide, biliverdin, and free iron [29]. HO-1 can have both antioxidative and anti-inflammatory effects, as biliverdin can be reduced to the antioxidant bilirubin, by biliverdin reductase, and carbon monoxide can have anti-inflammatory and apoptotic effects [30,31]. HO-1 mRNA and protein expression are commonly up-regulated following oxidative stress and cellular injury [32], and Nrf2 has been shown to directly regulate HO-1 promoter activity [14,33]. However, other mechanisms of transcriptional regulation are known to exist for HO-1 [34]. For example, Bach1 is a heme-sensing protein that binds to and inhibits Maf proteins, the crucial heterodimer partner for Nrf2 to bind to an ARE [26,35]. In addition, we and others have shown that NF-κB and AP-1 can also regulate HO-1 transcription. Moreover, Kirino and colleagues have shown that the pro-inflammatory cytokine tumor necrosis factor-α (TNF) can induce down regulation of HO-1 in human monocytes by promoting the degradation of HO-1 mRNA [36]. Therefore, HO-1 is regulated by multiple mechanisms in addition to Nrf2, suggesting that other Nrf2-target genes could be quantified as markers for Nrf2 activation.

NQO1, also known as DT-diaphorase, is a flavoprotein that is known to catalyze two electron reduction of a broad range of substrates [37]. It is also regarded as a prototypical Nrf2-target gene. NQO1 is critical for cytoprotection against many highly reactive and potentially damaging quinines. This is highlighted in Nrf2 null mice which have reduced constitutive expression and activity of NQO1 in liver, forestomach and small intestine [38]. Furthermore, a non-synonymous mutation in the NQO1 gene, leading to absence of enzyme activity, has been associated with an increased risk of myeloid leukemias and other types of blood dyscrasia in workers exposed to benzene [39].

GCL is an enzyme composed of two subunits: a modifier subunit (GCLM) and a catalytic subunit (GCLC), both of which contain ARE sequences in their promoters [11,12]. GCL performs the rate-limiting and ATP-dependent step in glutathione (GSH) synthesis by catalyzing the formation of γ-glutamyl cysteine from glutamine and cysteine. GSH maintains intracellular redox balance and protects against oxidative stress. Additionally, GSH can detoxify chemicals through direct binding or enzymatic conjugation by glutathione-S-transferase (GST) enzymes [40,41]. GSH also plays an important role in free radical scavenging.

GST catalyze the nucleophilic attack by reduced GSH on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom, often resulting in detoxification. GST mRNA and protein expression are decreased in Nrf2-null mice, and Nrf2 is required for GST induction [42]. Moreover, the mRNA expression of GST is markedly increased in Keap1-null mice [43].

MRP are ATP-dependent efflux transporters that export a wide-range of substrates, but especially glutathione, glucuronide, and sulfate conjugates. Four (MRP2, 3, 4, and 6) of the eight MRP expressed in mice are expressed in liver. Furthermore, Mrp2, 3, 4, and 6 in liver are induced by several Nrf2 activators, namely BHA, oltipraz, and ethoxyquin [44,45] However, Keap1-knockdown mice which have a whole body partial knockdown of Keap1 have only a modest induction of MRP2 mRNA expression in liver and no increase in hepatic MRP2 protein expression [43]. This observation, coupled with another study that demonstrated the capability of other transcription factors, such as NF-κB, contribute to MRP regulation, suggests that other regulatory mechanisms might control MRP induction [46,47].

This is by no means anywhere near a full list of the genes that are regulated by Nrf2, indeed there are over a hundred genes regulated by this process and we cannot possibly include all in this review. However, what this section has shown is the nature of the genes that are regulated by this signaling mechanism, and how cancer cells might be able to manipulate the function of these genes in order to provide their anti-apoptotic advantage during chemotherapy treatment.

CML is a hematopoietic stem cell disease defined by the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation t(9;22) [63]. The resulting fusion of the c-Abl- and BCR genes generates the BCR/Abl oncogene, which encodes a 210 kDa oncoprotein, BCR/Abl, exhibiting constitutive Abelson tyrosine kinase (TK) activity [64]. An important mechanism in the pathogenesis of CML appears to be enhanced survival and consequent accumulation of leukemic cells [65]. Correspondingly, BCR/Abl has been described to promote expression of a number of antiapoptotic molecules in leukemic cells [66].

During the past few years, imatinib (Gleevec) has become frontline therapy against CML and has been shown to be superior in producing complete cytogenetic and molecular responses compared with other drugs [67]. However, resistance against imatinib can occur during therapy with imatinib, particularly in accelerated phase (AP) and blast phase (BP) CML, as well as in acute lymphoblastic leukemia (ALL), and can represent a serious clinical problem [68,69].

With regards to Nrf2 in CML, Bonovolias et al. have recently shown that Nrf2-regulated genes can counteract imatinib-induced apoptosis [60], Moreover a number of studies have revealed that HO-1 provides resistance to imatinib and other CML targeted drugs and that silencing HO-1 removes the resistant nature of these cells [70,71]. Moreover, the Nrf2-regulated genes NQO1 and glutathione have both been linked to providing protection against apoptosis in human CML [72,73]. These data suggest that Nrf2 and its associated cytoprotective genes can protect CML from chemotherapy-induced apoptosis.

AML is one the most common malignant hematopoietic disorders in adults. It comprises a heterogenous group of clonal disorders of hematopoietic progenitors, showing genetic instability. Although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive leukemic stem cell population is likely to be drug-resistant. Moreover, in AML currently available drugs do not effectively distinguish between malignant stem cells and normal hematopoietic stem cells. Leukemic stem cells, which are quiescent or slowly cycling and therefore less sensitive to chemotherapy, are responsible for disease relapse and represent the target for future innovative therapies [74-76]. Furthermore, with 75% of patients diagnosed after the age of 60 and with current intensive chemotherapeutic strategies generally limited to a minority of younger, fitter patients there is a significant unmet need for better tolerated, more widely applicable and targeted anti-AML chemotherapy [77]. Our goal is to exploit unique properties of leukemic cells to induce apoptosis in the leukemic stem cell population, while sparing normal stem cells. One very important difference between normal and leukemic stem cell populations is constitutive NF-κB activation, which has therefore become a target for anticancer drug development [77,78].

NF-κB is an inducible transcription factor and central coordinator of immune responses. In addition to these functions, NF-κB signaling plays a critical role in cancer development and progression NF-κB pathway has been extensively studied in AML and been found to be constitutively activated in AML blast cells derived from patients [78,79]. Further, increased NF-κB activity has been strongly correlated with AML blast cell count [80]. Since then, several studies have explored targeting NF-κB activity in AML. For example, a study using high concentrations of an IKK2 (inhibitor of κB kinase-2) inhibitor showed induced apoptosis in three out of 15 AML samples [79]. The NF-κB pathway can also be inhibited indirectly by agents that target the proteasome, such as bortezomib and PR-171, but studies in AML show that bortezomib and PR-171 can induce only limited cell death [78]. The use of such NF-κB inhibitors in these studies has been disappointing, implying a limited role for targeting NF-κB transcription factor in AML. The other type of inhibitor of this pathway is targeted to blocking IκB kinase phosphorylation. This too has had limited success, which is highlighted in our studies using BAY-11-7082 (inhibitor of IκB phosphorylation), where AML-derived cell lines HL60, THP-1 and U937 revealed that inhibiting NF-κB had little effect on inducing apoptosis in these AML cells lines and primary samples [17,55]. Clearly other pathways must be targeted to discover more effective AML therapies.

To date, little is known regarding the role of Nrf2 in AML. Our own investigations have shown that in AML-derived cells, but not primary cells, HO-1 is up-regulated in response to TNF stimulation in conjunction with NF-κB inhibition. Furthermore, this induction of HO-1 protects AML cells from cell death signals [55]. The signaling machinery involved in regulating this response is Nrf2. HO-1 and Nrf2 may protect against the detrimental effects of inflammation and oxidative stress but may also help protect cancerous AML cells from TNF-mediated cell death, resulting in clinically devastating levels of apoptosis resistance. Moreover, our work and a study by a Japanese group have revealed that Nrf2 is constitutively active in some AML samples but not all suggesting that mutations may well be found in either Keap1 and/or Nrf2 in AML in the near future [61,81]. The study by Miyazaki et al. showed that constitutive Nrf2 induces HO-1 which provides protection for AML cells from chemotherapy-induced apoptosis [61] which will be discussed in more detail in the next section.

CLL is the most common form of leukemia in the Western world. Despite advances in our understanding of the biology of CLL, its current management includes monitoring without treatment of asymptomatic patients until disease progresses by clinical criteria. Improving the care of slowly progressing CLL will require the development of compounds with minimal or no toxicity. Recent studies have shown that drugs which induce reactive oxygen species are not as potent at inducing cytotoxicity of CLL, and since Nrf2 signaling pathway regulates the oxidative stress response [82], it would be fair to implicate Nrf2 in the protection of cells against drugs which induce reactive oxygen species. A study by Wu et al. showed that untreated CLL had elevated Nrf2 levels when compared with normal lymphocytes [58]. Moreover, 27 known electrophilic and antioxidant compounds with drug-like properties were tested in for cytotoxic effects on CLL and specifically on targeting Nrf2 signaling. The selected compounds were from five distinct structural classes; alpha-beta unsaturated carbonyls, isothiocyanates, sulfhydryl reactive metals, flavones, and polyphenols. There results showed that compounds containing alpha-beta unsaturated carbonyls, sulfhydryl reactive metals, and isothiocyanates are strong activators of Nrf2 in a reporter assay system and in primary human CLL based on increased expression of the Nrf2 target HO-1. Alpha-beta unsaturated carbonyl-containing compounds were selectively cytotoxic to CLL, and loss of the alpha-beta unsaturation abrogated Nrf2 activity and CLL toxicity. The alpha-beta unsaturated carbonyl containing compounds ethacrynic acid and parthenolide activated Nrf2 in normal peripheral blood mononuclear cells, but had a less potent effect in CLL cells. Furthermore, ethacrynic acid bound directly to the Nrf2-negative regulator Keap1 in CLL cells. These experiments document the presence of Nrf2 signaling in human CLL and suggest that altered Nrf2 responses may contribute to the observed selective cytotoxicity of electrophilic compounds in this disease.

In this section we have evaluated the existing data regarding Nrf2 and its associated genes protecting leukemia and lymphomas from undergoing apoptosis in response to endogenous signals as well as to chemotherapeutic drugs. We have hopefully demonstrated that new drugs or ways to manipulate the role of Nrf2 in these cancers are required.