Skin is the largest and one of the most complex organs in human body, accounting for almost 15% of total body weight. It serves as an important environmental interface and thus acts as a first line of defense against various environmental insults [1]. Skin is organized into three main layers, epidermis, dermis and subcutaneous layer. The epidermis, an outermost avascular layer, is formed by keratinocytes at the epidermal basal layer that differentiate into corneocytes at the outer layer of the epidermis. The dermis lies below the epidermis separated by a basement membrane and is composed mainly of fibroblasts. The primary function of skin is to constitute an efficient barrier to protect the organism both from water evaporation [2] and from damage, as such skin is exposed to many external and internal aggressors which can induce DNA damage, including ultraviolet radiations (UVR). The ultraviolet radiation component of sunlight is the most important environmental inducer of damage in the skin. Ultraviolet radiations can induce damage on DNA bases by direct absorption of photons resulting in the direct effect of cyclobutane pyrimidine dimers (CPD) or the 6-4 photoproducts formation both created by dimerization of contiguous pyrimidines on the DNA [2,3]. Ultra‐ violet radiation can also induces significant damage to skin cells through the generation of Reactive Oxygen Species (ROS) which produce secondary damage to DNA nucleobases and the sugar phosphate backbone [2]. Different forms of DNA damage can result from the type of ROS generated (singlet oxygen and hydroxyl radicals through the formation of superoxide radicals), different modifications are generated to DNA such as bulky (8-oxo- guanosine, as

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

guanine is the most easily oxidized base, Thymidine and Cytosine glycol) and non-bulky (cyclo purine and etheno adducts) base modifications, spontaneous hydrolysis of a normal or damaged nucleobase leading to an abasic site, (see review [4). Finally ROS may also generate other forms of DNA damage such as single strand breaks (SSB) or double strand breaks (DSB) when the free radical attack is located on the poly- deoxy- ribose chain [2]. In addition to ultraviolet radiation, pollution and cigarette smoke can also act as external aggressors and favor DNA damage onset by depleting intracellular anti-oxidant molecules such as glutathione and thus shifting the oxidative balance to favor oxidation by ROS. In addition to external aggression, cells are also subjected to internal aggression from ROS generated by oxidative metabolism or respiration as well as to the attack of genotoxic or photo-sensitizers coming from the diet [2].

The integrity of DNA is one of the key factors affecting the viability of healthy organisms, living cells have developed strategies not only to prevent DNA damage but also to minimize the impact of DNA damage by efficiently repairing any damaged DNA. In human cells, DNA is repaired by different mechanisms: Base Excision Repair (BER), Nucleotide Excision Repair (NER), Single and Double stranded Breaks Repair (SSBR and DSBR), Homologous Recombi‐ nation (HR) and Mismatched repair. Basically, DNA alterations without strand breaks are repaired mainly by excision repair mechanisms where the damaged bases are removed from the DNA molecule by excision and then replaced the right bases [2]. In the case of the Nu‐ cleotide Excision Repair (NER) an oligonucleotide fragment of approximately 25-30 nucleoti‐ des is removed around the damaged DNA and the gap generated in the DNA duplex is filled by DNA synthesis using the opposite, normal DNA strand as a template. To complete the process of NER, the last nucleotide incorporated is covalently joined to the extent DNA by ligation [5]. BER consists of four to five steps in which specific enzymes play a role: excision of the damaged base by a glycosylase, incision of the resulting abasic site, processing of the generated termini at the strand break, DNA synthesis and ligation [6,7]. A third mechanism called mismatched repair occurs when only one nucleotide mismatch appears in the DNA double chain. This mechanism is particularly effective for the repair of DNA error arising during replication due to the limited fidelity of the replicative machinery. Finally, DNA double strand breaks can be repaired by a specific process called homologous recombination and non homologous end joining [2,8].

The importance of the DNA repair process and its relevance in the skin physiology is apparent in genetic disorders affecting genes responsible for DNA repair. Xeroderma Pigmentosum (XP), Cockayne syndrome (CS) and Ataxia telangiectasia (AT) are genetic diseases resulting from rare autosomal recessive pathologies involving DNA repair enzymes that are deficient due to inactivating mutation in their genes [9,10,11]. These diseases are characterized at the level of the skin by extreme sensitivity to sunlight, resulting in sunburn, pigmentation changes, an early onset of the appearance of skin aging signs and a greatly elevated incidence of skin cancers in particular for XP disorder [12]. These changes can be explained by long lasting DNA damages that induces prolonged cellular inflammation through the activation of the NF-κB pathway [2,13,14,15,16] and an acquired immune deficiency [17] as well as rapid accumulation of mutation leading to cell apoptosis, senescence and cell tumorigenesis [18,19,20,21].

Humans share repair pathways with plants, particularly nucleotide excision repair (NER). NER is essential in removing major damage to DNA which interferes with the genetic code. Due to similarities in DNA damage and repair mechanisms in plants and humans, metabolites such as polyphenols produced by plants may provide beneficial effects in humans [22].

## **2. Photo protective mechanisms in plants**

guanine is the most easily oxidized base, Thymidine and Cytosine glycol) and non-bulky (cyclo purine and etheno adducts) base modifications, spontaneous hydrolysis of a normal or damaged nucleobase leading to an abasic site, (see review [4). Finally ROS may also generate other forms of DNA damage such as single strand breaks (SSB) or double strand breaks (DSB) when the free radical attack is located on the poly- deoxy- ribose chain [2]. In addition to ultraviolet radiation, pollution and cigarette smoke can also act as external aggressors and favor DNA damage onset by depleting intracellular anti-oxidant molecules such as glutathione and thus shifting the oxidative balance to favor oxidation by ROS. In addition to external aggression, cells are also subjected to internal aggression from ROS generated by oxidative metabolism or respiration as well as to the attack of genotoxic or photo-sensitizers coming

The integrity of DNA is one of the key factors affecting the viability of healthy organisms, living cells have developed strategies not only to prevent DNA damage but also to minimize the impact of DNA damage by efficiently repairing any damaged DNA. In human cells, DNA is repaired by different mechanisms: Base Excision Repair (BER), Nucleotide Excision Repair (NER), Single and Double stranded Breaks Repair (SSBR and DSBR), Homologous Recombi‐ nation (HR) and Mismatched repair. Basically, DNA alterations without strand breaks are repaired mainly by excision repair mechanisms where the damaged bases are removed from the DNA molecule by excision and then replaced the right bases [2]. In the case of the Nu‐ cleotide Excision Repair (NER) an oligonucleotide fragment of approximately 25-30 nucleoti‐ des is removed around the damaged DNA and the gap generated in the DNA duplex is filled by DNA synthesis using the opposite, normal DNA strand as a template. To complete the process of NER, the last nucleotide incorporated is covalently joined to the extent DNA by ligation [5]. BER consists of four to five steps in which specific enzymes play a role: excision of the damaged base by a glycosylase, incision of the resulting abasic site, processing of the generated termini at the strand break, DNA synthesis and ligation [6,7]. A third mechanism called mismatched repair occurs when only one nucleotide mismatch appears in the DNA double chain. This mechanism is particularly effective for the repair of DNA error arising during replication due to the limited fidelity of the replicative machinery. Finally, DNA double strand breaks can be repaired by a specific process called homologous recombination and non

The importance of the DNA repair process and its relevance in the skin physiology is apparent in genetic disorders affecting genes responsible for DNA repair. Xeroderma Pigmentosum (XP), Cockayne syndrome (CS) and Ataxia telangiectasia (AT) are genetic diseases resulting from rare autosomal recessive pathologies involving DNA repair enzymes that are deficient due to inactivating mutation in their genes [9,10,11]. These diseases are characterized at the level of the skin by extreme sensitivity to sunlight, resulting in sunburn, pigmentation changes, an early onset of the appearance of skin aging signs and a greatly elevated incidence of skin cancers in particular for XP disorder [12]. These changes can be explained by long lasting DNA damages that induces prolonged cellular inflammation through the activation of the NF-κB pathway [2,13,14,15,16] and an acquired immune deficiency [17] as well as rapid accumulation

of mutation leading to cell apoptosis, senescence and cell tumorigenesis [18,19,20,21].

from the diet [2].

316 Advances in DNA Repair

homologous end joining [2,8].

Sun light is a source of energy to sustain all types of life on earth including plants and animals. Sun light exposure specifically excessive sun light exposure can be harmful to plants and animals. Plants do not have the ability of movement therefore plants developed various mechanisms to protect them from harmful effects of sun light. Most harmful effects come from UVB part of sun light. It is known that excess UVB exposure adversely affects plant growth and development in many ways including nutrient uptake and photosynthesis rates [23]. At a cellular and molecular level DNA is the most important target for UV radiation specifically concerning are exposures from UV-B and UV-C regions of the spectrum [24]. The table below documents adverse effects of UV radiation on some additional cellular and molecular targets of plants. The adverse effects on a molecular level also alter the genetic makeup of cells by introducing mutations in DNA.

**Table 1.** Plants are adversely affected by excessive exposure of UV radiation

To adapt to the continuous insult from excessive sun light exposure, plants developed physical, enzymatic and non-enzymatic mechanisms to not only protect but also to repair the damage done from indiscriminate exposure of UV radiation. Some of the secondary metabolites for example polyphenols such as green tea polyphenols have been studied [37]. Green tea polyphenols plays protective role by mediating DNA repair and reduction in skin inflamma‐ tion. Polyphenols of various classes used topically [38 or consumed via diet as fruit and vegetables [39] helps to scavenge free radicals produced during exposure of light and also to mediate additional signaling pathways leading to ultimate damage of DNA at a molecular level.

## **3. Botanical extracts — Princess Tree as an antioxidant**

Paulownia tomentosa is commonly known as Princess Tree, Empress Tree, Royal Empress Tree, Royal Paulownia, Fox glove tree, Kiri (in Japan), PaoTong (China), and Odong-Namoo (Korea). Paulownia plants are well respected in Japan, China and most of East Asia for its tradition, uses and quality of wood. According to traditional literature flowers and leaves are cooked and consumed occasionally for the treatment of fever and pain, and skin ailments [40]. Recently the wood of Princess Tree has also been reported to possess anti-oxidant activity. The major polyphenol found in Princess Tree wood is Paulownin which belongs to a class of chemistry called lignan.

## **4. Phytochemical profile**

Paulownia plants are a rich source of phytochemistry documented by many studies and are reviewed comprehensively [41]. Expressed phytochemistry as a function of a part of the Paulownia tomentosa plant is shown in Table-1 below. Fruit, flower, and leave express specific chemistries of prenylated flavonoids, essential oil, and terpenoids resp. There are other examples of non-specific expression of flavonoids and phenolics by aerial parts and woody parts of the plant.

**Table 2.** Phytochemicals reported from parts of Paulownia tomentosa plant (derived from 2014 review cited above)

Essential oil from Paulownia flower is reported with identification of major components [42, e.g. benzyl alcohol (13.72%), phenol, 3,4-dimethoxy-methyl ester (3.64%), phenol, 2-me‐ thoxy-3-(2-popenyl)-methyl ester (6.24%), 1,2,4-Trimethoxybenzene (8.32%), tricosane (3.28%), and pentacosane (3.26%). A number of additional studies are also reported with similar chemical composition of Paulownia flower oil [43 and their anti-microbial activities. The heartwood of Paulownia is known to express Paulownin, Sesamin, lapachones, sterols, and naphoquinones.

## **5. Princess Tree reduces UV-induced reactive oxygen species and cellular inflammation**

#### **5.1. Free radicals & ROS**

mediate additional signaling pathways leading to ultimate damage of DNA at a molecular

Paulownia tomentosa is commonly known as Princess Tree, Empress Tree, Royal Empress Tree, Royal Paulownia, Fox glove tree, Kiri (in Japan), PaoTong (China), and Odong-Namoo (Korea). Paulownia plants are well respected in Japan, China and most of East Asia for its tradition, uses and quality of wood. According to traditional literature flowers and leaves are cooked and consumed occasionally for the treatment of fever and pain, and skin ailments [40]. Recently the wood of Princess Tree has also been reported to possess anti-oxidant activity. The major polyphenol found in Princess Tree wood is Paulownin which belongs to a class of

Paulownia plants are a rich source of phytochemistry documented by many studies and are reviewed comprehensively [41]. Expressed phytochemistry as a function of a part of the Paulownia tomentosa plant is shown in Table-1 below. Fruit, flower, and leave express specific chemistries of prenylated flavonoids, essential oil, and terpenoids resp. There are other examples of non-specific expression of flavonoids and phenolics by aerial parts and woody

Geranylated Flavonoids Flower, Fruit Mimulone, Diplacone, Diplacol, Schizolaenone C,

Phenolic glycosides Bark, Stem, wood Syringin, coniferin, Acteoside, Campenoside, Ilicifolioside,

Lignan / Phenolics Wood, Leaves, bark, FlowersPaulownin, Sesamin, Piperitol, Vanillic acid, gallic acid,

Terpenoids Leaves Iridoids: Paulownioside, catalpol, aucubin, tomentoside

**Table 2.** Phytochemicals reported from parts of Paulownia tomentosa plant (derived from 2014 review cited above)

Apigenin, kaempferol, Luteolin, Quercetin, Catechin,

Prokinawan, Tomentodiplacone, Tomentin

Naringenin, Taxifolin.

Isoverbascoside, Cistanoside

cinnamic acid, coumaric acid

**3. Botanical extracts — Princess Tree as an antioxidant**

level.

318 Advances in DNA Repair

chemistry called lignan.

parts of the plant.

**4. Phytochemical profile**

**Class of Chemistry Part of the plant Select Examples**

Prenylated Flavonoids Fruit Prenylated taxifolin

Quinones Stem, Bark Furanoquionones, plumbagin

Essential Oil Flower Cosanes, benzyl alcohol

Glycerides Leaves exudates Acyl glycerols

Flavonoids Leaves, bark, fruit, Stem,

Flowers

Antioxidants primarily mitigate the negative effect of free radicals through their radicalscavenging ability. These antioxidants stabilize radicals by donating electrons and thus preventing oxidation of DNA or other cellular components. While the body is equipped with its own defense system against reactive oxygen species (ROS) and other free radicals produced in the body, it also relies on external (exogenous) antioxidants including those contained in food. As environmental conditions lead to premature aging, a search for a suitable antioxidant product is vital [22].

Free radicals cause damage in the body because of their instability and high reactivity. ROS are of particular interest. During aerobic respiration, mitochondrial electron transport results in the formation of a ROS (superoxide) as a by-product. Solar UV radiation also leads to formation of ROS. Oxygen is particularly vulnerable to radical formation due to its electronic configuration with two valence shell unpaired electrons. Thus, there are several types of ROS including superoxide, hydrogen peroxide, nitric oxide, and hydroxyl radical. Free radicals of other atomic species specifically nitrogen are also formed within the body [22].

ROS can potentially react with other cellular entities including DNA which can lead to DNA modification and ultimately bodily harm. The guanine base in DNA is particularly susceptible to attach by ROS formed by solar UV radiation. Oxidation reactions which modify the guanine base may also lead to single-strand breaks in DNA [44]

While the effects of oxidative stress on the body vary according to type and duration, cells often halt division (enter G0 phase) and may even undergo apoptosis under severe stress. The general response to oxidative stress is cell cycle arrest through the expression of various inhibitor proteins (such as p21). Nevertheless, ROS also serve useful roles within the body including intracellular and intercellular communication [44].

#### **5.2. Antioxidants combat oxidative stress**

While broad-spectrum sunscreen which absorbs and reflects harmful solar radiation remains the most effective protection against immediate solar UV damage (which result in CPD formation), antioxidants are crucial in combating oxidative stress caused by ROS. Skin's antioxidant system consists of vitamins (vitamins C and E), enzymes (catalase and superoxide dismutase), glutathione, and coenzyme Q10 (CoQ10). As these antioxidants perform their protective actions and are degraded by ROS, they are reactivated by other antioxidants. Because several types of ROS may be formed through environmental insult, several types of antioxidants are produced in the skin. Thus antioxidants come in various forms (vitamins, enzymes, etc.) and may be either lipophilic or hydrophilic to function in a variety of areas [22].

During tissue damage and the subsequent inflammation, a number of mediators are released which have been shown to modulate DNA repair. The activation of the Melanocortin Receptor 1 (MCR1) by either its natural ligand, the α-Melanocyte stimulating Hormone αMSH or synthetic analogs [20,21] can enhance the DNA repair activity in cells. Also two interleukins (IL), IL12 and IL23 known to display anti-tumor activity [45,46,47,48] have been shown to accelerate the repair of UVB induced CPDs [2]. Activation of detoxifying mechanisms such as the NRf2 pathway may enhance also DNA repair [49]. Finally mono- and poly- ubiquitilation as well as sumoylation play an important role in the regulation of DNA repair (see review [50]). Thus inflammatory mediators can directly affect the DNA repair process and therefore could be regulatory factors either enhancing or repressing DNA repair. Recent studies have identi‐ fied that the NF-κB pathway, which is a key regulator in the expression of inflammatory proteins, may be an important mediator in DNA damage and the subsequent repair [2].

Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their anti-oxidant and for skin protective effects. Preincubation with Princess Tree wood extract at concentrations from 0.1% to 5% significantly attenuated hydrogen peroxide pro‐ duction in a dose-dependent manner (Figure 1A, \*P<0.05 compared with UV exposed vehicle treated epidermal equivalents). UV-induced hydrogen peroxide formation was determined using a modification of the method of Martin et al.,. Through free radicals scavenging activity Princess Tree wood extract may protect skin from oxidative stress that could result in DNA damage.

Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their anti-inflammatory activity and for skin protective effects. Preincubation with Princess Tree wood extract at concentrations from 0.1% to 5% significantly inhibited pro-inflammatory cytokine release. In the study shown in Figure 1B and 1C, Epidermal equivalents were topically treated (2mg/cm2 ) with Princess tree extracts in 70% ethanol/30% propylene glycol vehicle 2 hours before exposure to solar ultraviolet light (1000W-Oriel solar simulator equipped with a 1-mm Schott WG 320 filter; UV dose applied: 70 kJ/m2 as measured at 360nm). Supernatants were analyzed after 24 hours for IL-1A and IL-8 cytokine release using commercially available kits. These results clearly demonstrate that Princess Tree wood extract can reduce the skin inflammation and damage resulting from UV exposure.

#### **5.3. NF-κB Signal transduction**

Nuclear factor-κB (NF-κB) consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival [52. The NF-κB family of transcription factors shares a high-conserved sequence of amino acids within their

Paulownia tomentosa (Princess Tree) Extract Reduces DNA Damage and Induces DNA Repair Processes in Skin Cells http://dx.doi.org/10.5772/60005 321

formation), antioxidants are crucial in combating oxidative stress caused by ROS. Skin's antioxidant system consists of vitamins (vitamins C and E), enzymes (catalase and superoxide dismutase), glutathione, and coenzyme Q10 (CoQ10). As these antioxidants perform their protective actions and are degraded by ROS, they are reactivated by other antioxidants. Because several types of ROS may be formed through environmental insult, several types of antioxidants are produced in the skin. Thus antioxidants come in various forms (vitamins, enzymes, etc.) and may be either lipophilic or hydrophilic to function in a variety of areas [22]. During tissue damage and the subsequent inflammation, a number of mediators are released which have been shown to modulate DNA repair. The activation of the Melanocortin Receptor 1 (MCR1) by either its natural ligand, the α-Melanocyte stimulating Hormone αMSH or synthetic analogs [20,21] can enhance the DNA repair activity in cells. Also two interleukins (IL), IL12 and IL23 known to display anti-tumor activity [45,46,47,48] have been shown to accelerate the repair of UVB induced CPDs [2]. Activation of detoxifying mechanisms such as the NRf2 pathway may enhance also DNA repair [49]. Finally mono- and poly- ubiquitilation as well as sumoylation play an important role in the regulation of DNA repair (see review [50]). Thus inflammatory mediators can directly affect the DNA repair process and therefore could be regulatory factors either enhancing or repressing DNA repair. Recent studies have identi‐ fied that the NF-κB pathway, which is a key regulator in the expression of inflammatory proteins, may be an important mediator in DNA damage and the subsequent repair [2].

Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their anti-oxidant and for skin protective effects. Preincubation with Princess Tree wood extract at concentrations from 0.1% to 5% significantly attenuated hydrogen peroxide pro‐ duction in a dose-dependent manner (Figure 1A, \*P<0.05 compared with UV exposed vehicle treated epidermal equivalents). UV-induced hydrogen peroxide formation was determined using a modification of the method of Martin et al.,. Through free radicals scavenging activity Princess Tree wood extract may protect skin from oxidative stress that could result in DNA

Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their anti-inflammatory activity and for skin protective effects. Preincubation with Princess Tree wood extract at concentrations from 0.1% to 5% significantly inhibited pro-inflammatory cytokine release. In the study shown in Figure 1B and 1C, Epidermal equivalents were topically

hours before exposure to solar ultraviolet light (1000W-Oriel solar simulator equipped with a 1-mm Schott WG 320 filter; UV dose applied: 70 kJ/m2 as measured at 360nm). Supernatants were analyzed after 24 hours for IL-1A and IL-8 cytokine release using commercially available kits. These results clearly demonstrate that Princess Tree wood extract can reduce the skin

Nuclear factor-κB (NF-κB) consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival [52. The NF-κB family of transcription factors shares a high-conserved sequence of amino acids within their

inflammation and damage resulting from UV exposure.

) with Princess tree extracts in 70% ethanol/30% propylene glycol vehicle 2

damage.

320 Advances in DNA Repair

treated (2mg/cm2

**5.3. NF-κB Signal transduction**

**Figure 1.** (a). Princess tree mitigates UV-induced ROS (b). Princess tree inhibits UV-induced pro-inflammatory media‐ tor IL-8 (c). Princess tree inhibits UV-induced pro-inflammatory mediator IL-1α

*amino terminus,* which contains a nuclear localization sequence that is involved in the dimeri‐ zation with sequence-specific DNA binding and with the inhibitory IκB proteins [2].

In unstimulated cells, NF-κB family proteins exists as heterodimers or homodimers that are sequestered in the cytoplasm in an inactive form by virtue of their association with a member of the IκB family of inhibitory proteins, most notably IκBα, IκBβ and IκBγ [2,53,54]. About 200 extracellular signals can lead to activation through the dissociation of NF-κB from the IκB proteins. These activating signals include viral and bacterial products, oxidative stress, proinflammatory cytokines including IL-1 and TNF-α, and phorbol esters [2,55,56,57,58,59]. Ultraviolet (UV) radiation from sunlight induces IL-1 and TNF-α and creates reactive oxygen species that then leads to NF-κB-mediated inflammation [2,60,61]. The kinase activity of IκK phosphorylates two serine residues (Ser32 and Ser36) on IκB proteins, which results in the ubiquitination and degradation of IκB by the proteasome. The degradation of IκB reveals the nuclear localization sequence of NF-κB [53,54]. Free NF-κB can then translocate to the nucleus and bind to a NF-κB *consensus* sequence present within the promoter region of target genes, thereby upregulating the expression of hundreds of genes, including cytokines (IL-1, -2, -6, etc.) [2], immunoreceptors (immunoglobin kappa light chain, MHC class I, etc.), cellular adhesion molecules (ICAM-1, VCAM-1, ELAM-1), and many others [59].

Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their NF-B activity. Preincubation with **Figure 2.** Princess tree inhibited NF-κB promoter activity

Princess Tree wood extract at concentrations from 0.001% to 0.01% significantly inhibited NF-B activation. In the study shown in Figure 2, a cell line expressing a NF-B promoter gene and internal control Renilla luciferase reporter gene were treated with the indicated doses of Princess tree and stimulated with Tumor Necrosis Factor-α (TNFα). Cell lysates were analyzed using Dual-Luciferase Reporter System. These results establish that Princess Tree wood extract can reduce the activation of NF-B and thus may reduce the cellular damage resulting from UV exposure. **NF-B and DNA Damage** The NF-B pathway has been shown to be regulated by ionizing radiation at both the mRNA and protein levels by Brach et al., who demonstrated that NF-B transcripts were transiently increased after irradiation, which was preceded by enhanced DNA binding activity of this transcription factor [62. Nuclear DNA double strand breaks (DSBs) are one of the most potent DNA damage signals Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their NF-κB activity. Preincubation with Princess Tree wood extract at concentrations from 0.001% to 0.01% significantly inhibited NF-κB activation. In the study shown in Figure 2, a cell line expressing a NF-κB promoter gene and internal control Renilla luciferase reporter gene were treated with the indicated doses of Princess tree and stimulated with Tumor Necrosis Factor-α (TNFα). Cell lysates were analyzed using Dual-Luciferase Reporter System. These results establish that Princess Tree wood extract can reduce the activation of NF-κB and thus may reduce the cellular damage resulting from UV exposure.

#### (IB) kinase (IKK) complex and IBa degradation [15. NF-B can be activated by Topoisomerase inhibitors (such as camptothecin) **5.4. NF-κB and DNA damage**

damage[2].

**NF-B and the DNA Repair Process**

topoisomerase inhibitors was described to be dependent on the zinc finger domain in NF-B essential modulator (NEMO) [50. DSBs can trigger two independent signaling cascades that eventually lead to the induction of NF-B via NEMO [61. In one case, DSBs can activate ATM, which in turn can bind to and phosphorylate NEMO. In a parallel cascade, the p53-induced protein with a The NF-κB pathway has been shown to be regulated by ionizing radiation at both the mRNA and protein levels by Brach et al., who demonstrated that NF-κB transcripts were transiently

to activate NF-B[2]. This process can occur within 1–2 h after break induction through activation of the canonical inhibitor of B

potentially via the generation of double strand breaks as well [16*.* Furthermore activation of IKK following treatment with

death domain (PIDD) translocates to the nucleus leading to the SUMOylation of NEMO. Consequently, the resulting activation of NF-B favors cell survival by turning on the transcription of several anti-apoptotic gene[2]s. In response to DSB, PIDD as well as ATM are capable of initiating cascades leading to pro- or antiapoptotic signals, NF-B presumably being a part of the pro-survival cascade [61. Miyamoto et al., have summarized this model of NF-B activation by DNA damage as a 'two signal' model as it requires coincident NEMO SUMOylation and ATM activation by double strand breaks to permit robust NF-B activation [15. Taken together these findings suggest that NF-B may be both have both causal and effector roles in the development of DNA

Although the mechanisms by which NF-B affects DNA damage are not fully established, one possibility is that NF-B may either

increased after irradiation, which was preceded by enhanced DNA binding activity of this transcription factor [62]. Nuclear DNA double strand breaks (DSBs) are one of the most potent DNA damage signals to activate NF-κB [2]. This process can occur within 1–2 h after break induction through activation of the canonical inhibitor of κB (IκB) kinase (IKK) complex and IκBa degradation [15]. NF-κB can be activated by Topoisomerase inhibitors (such as campto‐ thecin) potentially via the generation of double strand breaks as well [16]*.* Furthermore activation of IKK following treatment with topoisomerase inhibitors was described to be dependent on the zinc finger domain in NF-κB essential modulator (NEMO) [50]. DSBs can trigger two independent signaling cascades that eventually lead to the induction of NF-κB via NEMO [61]. In one case, DSBs can activate ATM, which in turn can bind to and phosphorylate NEMO. In a parallel cascade, the p53-induced protein with a death domain (PIDD) translocates to the nucleus leading to the SUMOylation of NEMO. Consequently, the resulting activation of NF-κB favors cell survival by turning on the transcription of several anti-apoptotic gene [2]s. In response to DSB, PIDD as well as ATM are capable of initiating cascades leading to pro- or antiapoptotic signals, NF-κB presumably being a part of the pro-survival cascade [61]. Miyamoto et al., have summarized this model of NF-κB activation by DNA damage as a 'two signal' model as it requires coincident NEMO SUMOylation and ATM activation by double strand breaks to permit robust NF-κB activation [15]. Taken together these findings suggest that NF-κB may be both have both causal and effector roles in the development of DNA damage [2].

#### **5.5. NF-κB and the DNA repair process**

In unstimulated cells, NF-κB family proteins exists as heterodimers or homodimers that are sequestered in the cytoplasm in an inactive form by virtue of their association with a member of the IκB family of inhibitory proteins, most notably IκBα, IκBβ and IκBγ [2,53,54]. About 200 extracellular signals can lead to activation through the dissociation of NF-κB from the IκB proteins. These activating signals include viral and bacterial products, oxidative stress, proinflammatory cytokines including IL-1 and TNF-α, and phorbol esters [2,55,56,57,58,59]. Ultraviolet (UV) radiation from sunlight induces IL-1 and TNF-α and creates reactive oxygen species that then leads to NF-κB-mediated inflammation [2,60,61]. The kinase activity of IκK phosphorylates two serine residues (Ser32 and Ser36) on IκB proteins, which results in the ubiquitination and degradation of IκB by the proteasome. The degradation of IκB reveals the nuclear localization sequence of NF-κB [53,54]. Free NF-κB can then translocate to the nucleus and bind to a NF-κB *consensus* sequence present within the promoter region of target genes, thereby upregulating the expression of hundreds of genes, including cytokines (IL-1, -2, -6, etc.) [2], immunoreceptors (immunoglobin kappa light chain, MHC class I, etc.), cellular

> Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their NF-B activity. Preincubation with Princess Tree wood extract at concentrations from 0.001% to 0.01% significantly inhibited NF-B activation. In the study shown in Figure 2, a cell line expressing a NF-B promoter gene and internal control Renilla luciferase reporter gene were treated with the indicated doses of Princess tree and stimulated with Tumor Necrosis Factor-α (TNFα). Cell lysates were analyzed using Dual-Luciferase Reporter System. These results establish that Princess Tree wood extract can reduce the activation of NF-B and thus

Paulownin and Paulownin rich extracts from wood of Paulownia tomentosa were studied for their NF-κB activity. Preincubation with Princess Tree wood extract at concentrations from 0.001% to 0.01% significantly inhibited NF-κB activation. In the study shown in Figure 2, a cell line expressing a NF-κB promoter gene and internal control Renilla luciferase reporter gene were treated with the indicated doses of Princess tree and stimulated with Tumor Necrosis Factor-α (TNFα). Cell lysates were analyzed using Dual-Luciferase Reporter System. These results establish that Princess Tree wood extract can reduce the activation of NF-κB and thus

The NF-κB pathway has been shown to be regulated by ionizing radiation at both the mRNA and protein levels by Brach et al., who demonstrated that NF-κB transcripts were transiently

**TNF-α stimulated**

The NF-B pathway has been shown to be regulated by ionizing radiation at both the mRNA and protein levels by Brach et al., who demonstrated that NF-B transcripts were transiently increased after irradiation, which was preceded by enhanced DNA binding activity of this transcription factor [62. Nuclear DNA double strand breaks (DSBs) are one of the most potent DNA damage signals to activate NF-B[2]. This process can occur within 1–2 h after break induction through activation of the canonical inhibitor of B (IB) kinase (IKK) complex and IBa degradation [15. NF-B can be activated by Topoisomerase inhibitors (such as camptothecin) potentially via the generation of double strand breaks as well [16*.* Furthermore activation of IKK following treatment with topoisomerase inhibitors was described to be dependent on the zinc finger domain in NF-B essential modulator (NEMO) [50. DSBs can trigger two independent signaling cascades that eventually lead to the induction of NF-B via NEMO [61. In one case, DSBs can activate ATM, which in turn can bind to and phosphorylate NEMO. In a parallel cascade, the p53-induced protein with a death domain (PIDD) translocates to the nucleus leading to the SUMOylation of NEMO. Consequently, the resulting activation of NF-B favors cell survival by turning on the transcription of several anti-apoptotic gene[2]s. In response to DSB, PIDD as well as ATM are capable of initiating cascades leading to pro- or antiapoptotic signals, NF-B presumably being a part of the pro-survival cascade [61. Miyamoto et al., have summarized this model of NF-B activation by DNA damage as a 'two signal' model as it requires coincident NEMO SUMOylation and ATM activation by double strand breaks to permit robust NF-B activation [15. Taken together these findings suggest that NF-B may be both have both causal and effector roles in the development of DNA

Although the mechanisms by which NF-B affects DNA damage are not fully established, one possibility is that NF-B may either

adhesion molecules (ICAM-1, VCAM-1, ELAM-1), and many others [59].

may reduce the cellular damage resulting from UV exposure.

may reduce the cellular damage resulting from UV exposure.

**NF-B and DNA Damage**

**Figure 2.** Princess tree inhibited NF-κB promoter activity

damage[2].

**5.4. NF-κB and DNA damage**

322 Advances in DNA Repair

**NF-B and the DNA Repair Process**

Although the mechanisms by which NF-κB affects DNA damage are not fully establish‐ ed, one possibility is that NF-κB may either directly or indirectly regulate DNA repair processes in cells. Protecting cells from apoptotic cell death following DNA damage is one of the major ways that NF-κB activation regulates the DNA repair process [2]. Wang et al., have demonstrated that NF-κB functions as a positive modulator of cellular senescence, an intrinsic tumor suppression mechanism, by showing that human fibroblasts lacking NF-κB activity prematurely exit from senescence [63]. Others have shown that skin cells devoid of NF-κB activity exhibit deregulated growth correlating with impaired cell-cycle control [64,65]. It has been proposed that the role of NF-κB in cellular senescence could be cell type specific, differentially initiating senescence or acting further downstream in the DNA repair process to maintain the senescent state [2,63]. DNA damage caused by chemical genotox‐ ic agents, such as camptothecin, has been described to activate the Ataxia Telangiectasia-Mutated (ATM) kinase and NEMO (IκB kinase), leading to the inducing of NF-κB p50/p65 heterodimer [66]. In a parallel signaling pathway, ROS can be generated by genotoxic agents in sufficient quantities to activate the NF-κB pathway. ROS can also act as signaling molecules in immune responses, cell death and inflammation, where NF-κB is involved [66]. Depending on the relative degree of DNA damage, multiple mechanisms of NF-κB activation are engaged. Physical genotoxic agents such as UVA or hydrogen peroxide lead to extensive oxidative damage within the cytoplasm which can signal the activation of NFκB pathway in the absence of DNA damage [2].

Among the various types of DNA damage, repairing double strand breaks can be particularly challenging to cells [67,68], and may contribute to genomic instability associated with most cancers [68,69,70,71]. Wiesmuller et al., have shown that NF-κB is involved in double strand removal and repair via a stimulatory action on homologous repair, involving the targets ATM and the tumor suppressor gene BRCA2 [72]. NF-κB is known to bind to the BRCA2 promoter and activate BRCA2 gene expression [73]. The role of NF-κB in ATM function and DNA repair was demonstrated by Siervi et al., in T-cells where levels of ATM mRNA and protein were significantly reduced by NF-κB blockade [74]. Activation of NF-κB by ATM results in an antiapoptotic signal in the cells. Wiesmuller et al. have also described that NF-κB utilizes multiple mechanisms to enhance homologous recombination, including stimulation of the activity of CtIP–BRCA1 complexes to trigger DNA end processing, and upregulation of ATM and BRCA2 for strand transfer [72].

The nuclear factor p53 controls several physiological processes including DNA repair and cell cycle arrest. Cross-talk between NF-κB and p53 has been established by multiple groups ( [75,76]; see review [77]), including results that suggest NF-κB may have both anti- and proapoptotic roles. Only a limited number of studies have investigated the role of NF-κB in DNA damage and repair in skin cells (including: [64,65,78,79,80,81]). Evaluation of the p53-NFκB cross-talk by Puszynski et al. in HaCat keratinocytes cells showed that inactivation of NF-κB improved p53-mediated DNA repair and prevented arsenite-induced malignant transforma‐ tion of HaCaT cells [80]. Marwaha et al. have shown that in primary skin cells, such as dermal fibroblasts and keratinocytes, treatment with T-oligos led to the up-regulation and activation of p53, coinciding with decreased NF-κB DNA binding activity and inhibition of transcription from NF-κB-driven promoter constructs [79]. Thyss et al. have demonstrated that the sequen‐ tial activation of NF-κB, Egr-1 and Gadd45 cascade induces UVB-mediated cell death in epidermal cells [81], a process that was crucial in order to eradicate the cells that bear the risk of becoming tumorigenic. In HaCat keratinocytes, hydroxytyrosol (main component of olive oil shown to be an inhibitor of NF-κB), has been shown to significantly reduce the DNA strand breaks caused by UVB, and also attenuate the expression of p53 and NF-κB in a concentrationdependent manner [78].

#### **5.6. Princess Tree reduces DNA damage**

The skin is the largest organ protecting the body against external threats such as physical, environmental and biological insults. Among the harmful environmental factors, solar ultraviolet radiation (UVR) is the major one causing cellular and molecular modifications in the skin like photo-aging and eventually leading to genomic instability and cancer ( [3, 82]. UV-induced generation of Reactive Oxygen Species (ROS) such as O2 , H2O2, OH° in the skin, develops oxidative stress when their formation exceeds the antioxidant defense ability. UVR penetrates the skin, reaches the cells, and is absorbed by DNA, leading to the formation of photoproducts that inactivate the DNA functions. Oxidation of DNA can product different types of DNA damages: strand breaks, sister chromatid exchange, DNA-protein crosslinks, sugar damage, abasic sites and base modifications [83,84]; [4]. One of the major DNA oxidation products formed as a result of such damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). DNA damage by UVB irradiation results also from photochemical reactions consequent to direct absorption of photons by DNA bases [51]. The UV-induced DNA lesions that have been studied in most detail are the cyclobutane pyrimidine dimer (CPD) and the 6-4 pyrimi‐ dine–pyrimidone photoproduct (6-4PP) at adjacent pyrimidines [51,85,86].

Among the various types of DNA damage, repairing double strand breaks can be particularly challenging to cells [67,68], and may contribute to genomic instability associated with most cancers [68,69,70,71]. Wiesmuller et al., have shown that NF-κB is involved in double strand removal and repair via a stimulatory action on homologous repair, involving the targets ATM and the tumor suppressor gene BRCA2 [72]. NF-κB is known to bind to the BRCA2 promoter and activate BRCA2 gene expression [73]. The role of NF-κB in ATM function and DNA repair was demonstrated by Siervi et al., in T-cells where levels of ATM mRNA and protein were significantly reduced by NF-κB blockade [74]. Activation of NF-κB by ATM results in an antiapoptotic signal in the cells. Wiesmuller et al. have also described that NF-κB utilizes multiple mechanisms to enhance homologous recombination, including stimulation of the activity of CtIP–BRCA1 complexes to trigger DNA end processing, and upregulation of ATM and BRCA2

The nuclear factor p53 controls several physiological processes including DNA repair and cell cycle arrest. Cross-talk between NF-κB and p53 has been established by multiple groups ( [75,76]; see review [77]), including results that suggest NF-κB may have both anti- and proapoptotic roles. Only a limited number of studies have investigated the role of NF-κB in DNA damage and repair in skin cells (including: [64,65,78,79,80,81]). Evaluation of the p53-NFκB cross-talk by Puszynski et al. in HaCat keratinocytes cells showed that inactivation of NF-κB improved p53-mediated DNA repair and prevented arsenite-induced malignant transforma‐ tion of HaCaT cells [80]. Marwaha et al. have shown that in primary skin cells, such as dermal fibroblasts and keratinocytes, treatment with T-oligos led to the up-regulation and activation of p53, coinciding with decreased NF-κB DNA binding activity and inhibition of transcription from NF-κB-driven promoter constructs [79]. Thyss et al. have demonstrated that the sequen‐ tial activation of NF-κB, Egr-1 and Gadd45 cascade induces UVB-mediated cell death in epidermal cells [81], a process that was crucial in order to eradicate the cells that bear the risk of becoming tumorigenic. In HaCat keratinocytes, hydroxytyrosol (main component of olive oil shown to be an inhibitor of NF-κB), has been shown to significantly reduce the DNA strand breaks caused by UVB, and also attenuate the expression of p53 and NF-κB in a concentration-

The skin is the largest organ protecting the body against external threats such as physical, environmental and biological insults. Among the harmful environmental factors, solar ultraviolet radiation (UVR) is the major one causing cellular and molecular modifications in the skin like photo-aging and eventually leading to genomic instability and cancer ( [3, 82]. UV-induced generation of Reactive Oxygen Species (ROS) such as O2 , H2O2, OH° in the skin, develops oxidative stress when their formation exceeds the antioxidant defense ability. UVR penetrates the skin, reaches the cells, and is absorbed by DNA, leading to the formation of photoproducts that inactivate the DNA functions. Oxidation of DNA can product different types of DNA damages: strand breaks, sister chromatid exchange, DNA-protein crosslinks, sugar damage, abasic sites and base modifications [83,84]; [4]. One of the major DNA oxidation products formed as a result of such damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-

for strand transfer [72].

324 Advances in DNA Repair

dependent manner [78].

**5.6. Princess Tree reduces DNA damage**

**Figure 3.** (a). Treatment of primary human keratinocytes with Princess Tree increased repair of UV-induced DNA damage (b). Treatment of primary human keratinocytes with Princess Tree increased repair of UV-induced DNA dam‐ age.

Mammalian cells have evolved several DNA-repair pathways to remove all the categories of DNA base lesions, relying in particular on DNA excision mechanisms. One of these, nucleotide excision repair, removes bulky adducts and is thus an essential mechanism for correcting UV-induced DNA damage [87,88]. The base excision repair pathway corrects small base modifications such as oxidized and alkylated bases [88,89]. The importance of repair mechanisms is demonstrated by the hazardous consequences of genetic defects in DNA repair [88,90,91].

Princess Tree wood extract was investigated for the capacity to promote DNA repair after UV insults (6DEM) using Comet assay. After 1 hour treatment, both concentrations of Princess Tree (10 and 100 µg/ml) reduced significantly UV-induced DNA damage in Normal Human Epidermal Keratinocytes (NHEK) when compared to UV-irradiated control as shown by the fluorescent images and the quantification of the comet tail (Fig 3A and 3B, p<0.05). The Princess Tree wood extract treatment increased the DNA damage repair rate. Indeed, 4 hours were needed for the UV-irradiated control to reach the same level of DNA damage/repair compared to the Princess Tree wood extract treated conditions.

Princess Tree wood extract direct effects on mitigating DNA damage may be by an indirect mechanism, such as the inhibition of NFkB pathway known to be regulated by ionizing radiation at both the mRNA and protein levels [62]

## **6. Summary and conclusions**

#### **6.1. The use of botanical extracts for protection from DNA damage and DNA repair**

Skin is under continual assault from a variety of damaging environmental factors such as ultraviolet irradiation and atmospheric pollutants. As organisms age the cumulative damage exceeds the capacity of endogenous antioxidant defenses resulting in oxidative damage. Furthermore, during oxidative stress the elevation of NF-κB transcriptional activity may contribute to the decrease in DNA repair capacity of skin cells and thereby lead to the accumulation of DNA damage. Since NF-κB is activated by DNA damage, there is a potential for a vicious circle to take place as more NF-κB may decrease the capacity of the cell to repair damages and lead to a longer persistence of the DNA damage. Plants have adapted to chronic exposure to ultraviolet irradiation by producing phytochemicals which can mitigate reactive oxygen species and repair damaged DNA. Botanical extracts such as Princess Tree (Paulownia tomentosa) which can modulate the NF-κB pathway, a primary pathway linking inflammation and DNA damage, can prevent the deleterious effects of DNA damage in cells (Figure 4). Through the ability to scavenge free radicals, inhibit NFκB activation, reduce DNA damage and induce repair of damaged DNA, Princess Tree may protect skin from numerous external aggressions encountered daily and reduce the damage to oxidatively challenged skin.

Paulownia tomentosa (Princess Tree) Extract Reduces DNA Damage and Induces DNA Repair Processes in Skin Cells http://dx.doi.org/10.5772/60005 327

**Figure 4.** Proposed model showing the effects of Paulownia tomentosa on DNA damage and repair

### **Acknowledgements**

Mammalian cells have evolved several DNA-repair pathways to remove all the categories of DNA base lesions, relying in particular on DNA excision mechanisms. One of these, nucleotide excision repair, removes bulky adducts and is thus an essential mechanism for correcting UV-induced DNA damage [87,88]. The base excision repair pathway corrects small base modifications such as oxidized and alkylated bases [88,89]. The importance of repair mechanisms is demonstrated by the hazardous consequences of genetic defects in

Princess Tree wood extract was investigated for the capacity to promote DNA repair after UV insults (6DEM) using Comet assay. After 1 hour treatment, both concentrations of Princess Tree (10 and 100 µg/ml) reduced significantly UV-induced DNA damage in Normal Human Epidermal Keratinocytes (NHEK) when compared to UV-irradiated control as shown by the fluorescent images and the quantification of the comet tail (Fig 3A and 3B, p<0.05). The Princess Tree wood extract treatment increased the DNA damage repair rate. Indeed, 4 hours were needed for the UV-irradiated control to reach the same level of DNA

Princess Tree wood extract direct effects on mitigating DNA damage may be by an indirect mechanism, such as the inhibition of NFkB pathway known to be regulated by ionizing

damage/repair compared to the Princess Tree wood extract treated conditions.

**6.1. The use of botanical extracts for protection from DNA damage and DNA repair**

Skin is under continual assault from a variety of damaging environmental factors such as ultraviolet irradiation and atmospheric pollutants. As organisms age the cumulative damage exceeds the capacity of endogenous antioxidant defenses resulting in oxidative damage. Furthermore, during oxidative stress the elevation of NF-κB transcriptional activity may contribute to the decrease in DNA repair capacity of skin cells and thereby lead to the accumulation of DNA damage. Since NF-κB is activated by DNA damage, there is a potential for a vicious circle to take place as more NF-κB may decrease the capacity of the cell to repair damages and lead to a longer persistence of the DNA damage. Plants have adapted to chronic exposure to ultraviolet irradiation by producing phytochemicals which can mitigate reactive oxygen species and repair damaged DNA. Botanical extracts such as Princess Tree (Paulownia tomentosa) which can modulate the NF-κB pathway, a primary pathway linking inflammation and DNA damage, can prevent the deleterious effects of DNA damage in cells (Figure 4). Through the ability to scavenge free radicals, inhibit NFκB activation, reduce DNA damage and induce repair of damaged DNA, Princess Tree may protect skin from numerous external aggressions encountered daily and reduce the damage

radiation at both the mRNA and protein levels [62]

**6. Summary and conclusions**

to oxidatively challenged skin.

DNA repair [88,90,91].

326 Advances in DNA Repair

The authors would like to thank José Serrano (Johnson and Johnson) for his technical assistance with the Comet assay, Thierry Oddos (Johnson and Johnson) for discussion on DNA repair, and Michelle Garay (Johnson and Johnson) for technical assistance on oxidative stress measurements.

#### **Author details**

Simarna Kaur, Heng Kuan Wong, Michael D. Southall and Khalid Mahmood

Johnson & Johnson Skin Research Center, CPPW, a division of Johnson & Johnson Consumer Companies, Inc. Skillman, New Jersey and Val de Reuil, USA

Parts of this chapter are reproduced from the authors' previous publications [2, 22].

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## **Chapter 11**

## **Targeting DNA Repair Mechanisms to Treat Glioblastoma**

Johnathan E. Lawrence, Cathy E. Bammert, Robert J. Belton Jr., Richard A. Rovin and Robert J. Winn

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59632

## **1. Introduction**

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The current standard of care for the treatment of glioblastoma multiforme (GBM) includes the surgical resection of the tumor in combination with ionizing radiation and the DNA alkylating agent Temozolomide (TMZ). The introduction of TMZ into clinical use has improved patient outcomes [1, 2]. Stupp et al. showed that the addition of TMZ to radiotherapy lengthened the median survival in patients with GBM from 12.1 months to 14.6 months [2]. TMZ exerts an effect upon GBM cells by preferentially damaging the DNA of the rapidly growing tumor cells, ultimately resulting in their death. While the combined use of TMZ and ionizing radiation can increase overall survival, the long-term survival for GBM patients is still poor [3]. What has recently become apparent is that GBM tumors can develop several forms of resistance to the DNA damage-induced cell death caused by radiotherapy and TMZ treatments. In this way, GBM tumors can survive and generate new tumors when they should otherwise not survive. This review will discuss mechanisms of resistance to DNA damage-induced cell death in GBM tumors and will outline some DNA repair functions that can be targeted to potentially improve treatment outcomes.

Maintaining the integrity of the genome is essential for the health and survival of multicellular organisms. The continuous exposure of cellular DNA to potentially harmful environmental and internal insults necessitates redundant and overlapping DNA repair mechanisms. Several excellent reviews have extensively described the wide variety of DNA repair mechanisms used by cells in response to DNA damage [4-6]. Damage to DNA can result in cell cycle arrest to allow for DNA repair mechanisms to occur, or can stall replication forks during DNA repli‐

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

cation causing senescence. Proliferating cells, like those in GBM tumors, are affected to a greater extent than quiescent cells following DNA damage, causing the cells to arrest at particular points within the cell cycle [7]. However, cancers such as GBMs are quite adept at repairing the DNA damage or over-riding the cell cycle checkpoints to allow cell proliferation to continue despite the damage.

GBM tumors respond to DNA damage induced by ionizing radiation and TMZ treatment through increased expression of DNA repair enzymes, including the proteins O-6-methyl‐ guanine-DNA methyltransferase (MGMT) and Poly (ADP-ribose) Polymerase 1 (PARP-1) [8]. Furthermore, tumors are able to eliminate chemotherapeutic compounds from cells through the increased expression and activity of ABC transporters, specifically ABC-1 [9]. Compound‐ ing this issue is the growing body of evidence indicating that a small population of slowgrowing cancer stem cells reside within the GBM tumor (also called glioma-initiating cells) and are responsible for the subsequent recurrence of GBM tumors [10, 11]. Glioma initiating cells are particularly resistant to standard treatments, in part through the elevated expression of enzymes responsible for repair of DNA damage [12, 13]. Therefore, successful destruction of GBM tumors may require a combined approach utilizing standard treatments in combina‐ tion with inhibition of DNA repair pathways. This approach to cancer treatment, called synthetic lethality, preferentially affects cancer cells by inhibiting several molecular processes necessary for tumor survival without significantly affecting normal tissues [14, 15]. This treatment approach utilizing the DNA repair enzymes MGMT and PARP-1 have been a focus of the research conducted at the Upper Michigan Brain Tumor Center, and this review will be supplemented with findings from our laboratory.

*Temozolomide*. TMZ is easily absorbed after oral administration, readily crosses the blood-brain barrier and is better tolerated than its parent compound, mitozolomide. TMZ is an imidazo‐ tetrazine prodrug that is converted to a compound, 5-(3-methyltriazen-1-yl)imidazole-4 carboximide (MTIC), capable of alkylating DNA and displays antitumor activity in a variety of cancer types [16]. Spontaneous conversion of TMZ to MTIC is pH dependent and is a chemically controlled reaction [1]. MTIC's most common sites of methylation are at the N7 position of guanine followed by the N3 position of adenine. The N7-methylguanine is stable and makes up 80-85% of all alkyl adducts, whereas, N3-methyladenine is readily hydrolyzed and comprises only 8-18% of adducts [8, 17]. The O6 position of guanine only makes up 5% of lesions but is the most stable of the three and persists in the DNA in the absence of MGMT enzyme activity [8, 17]. O6-methylguanine is considered to be the most lethal of the alkyl adducts. For a visual representation of these common adducts, see Figure 1.

*O-6-Methylguanine-DNA Methyltransferase*. The MGMT enzyme reduces the cytotoxicity of O6 alkylating agents like TMZ by catalyzing the transfer of methyl groups from the O6 position of guanine to a cysteine residue within the active site of the MGMT enzyme [1]. The transfer of ethyl or alkyl groups to MGMT renders the MGMT enzyme inactive, leading to its degra‐ dation via the proteosomal pathway [18]. Therefore, continued MGMT function requires the de novo expression of MGMT protein in order to provide resistance to chemotherapeutic agents. Unfortunately, many GBM tumors exhibit increased MGMT expression, which reduces the effectiveness of alkylating agents such as TMZ. GBM tumors with hypermethylated MGMT promoter regions do not develop resistance to the drug indicating that reduced MGMT expression in tumors is a clinically relevant and potentially important cellular phenotype to consider during treatment.

cation causing senescence. Proliferating cells, like those in GBM tumors, are affected to a greater extent than quiescent cells following DNA damage, causing the cells to arrest at particular points within the cell cycle [7]. However, cancers such as GBMs are quite adept at repairing the DNA damage or over-riding the cell cycle checkpoints to allow cell proliferation

GBM tumors respond to DNA damage induced by ionizing radiation and TMZ treatment through increased expression of DNA repair enzymes, including the proteins O-6-methyl‐ guanine-DNA methyltransferase (MGMT) and Poly (ADP-ribose) Polymerase 1 (PARP-1) [8]. Furthermore, tumors are able to eliminate chemotherapeutic compounds from cells through the increased expression and activity of ABC transporters, specifically ABC-1 [9]. Compound‐ ing this issue is the growing body of evidence indicating that a small population of slowgrowing cancer stem cells reside within the GBM tumor (also called glioma-initiating cells) and are responsible for the subsequent recurrence of GBM tumors [10, 11]. Glioma initiating cells are particularly resistant to standard treatments, in part through the elevated expression of enzymes responsible for repair of DNA damage [12, 13]. Therefore, successful destruction of GBM tumors may require a combined approach utilizing standard treatments in combina‐ tion with inhibition of DNA repair pathways. This approach to cancer treatment, called synthetic lethality, preferentially affects cancer cells by inhibiting several molecular processes necessary for tumor survival without significantly affecting normal tissues [14, 15]. This treatment approach utilizing the DNA repair enzymes MGMT and PARP-1 have been a focus of the research conducted at the Upper Michigan Brain Tumor Center, and this review will be

*Temozolomide*. TMZ is easily absorbed after oral administration, readily crosses the blood-brain barrier and is better tolerated than its parent compound, mitozolomide. TMZ is an imidazo‐ tetrazine prodrug that is converted to a compound, 5-(3-methyltriazen-1-yl)imidazole-4 carboximide (MTIC), capable of alkylating DNA and displays antitumor activity in a variety of cancer types [16]. Spontaneous conversion of TMZ to MTIC is pH dependent and is a chemically controlled reaction [1]. MTIC's most common sites of methylation are at the N7 position of guanine followed by the N3 position of adenine. The N7-methylguanine is stable and makes up 80-85% of all alkyl adducts, whereas, N3-methyladenine is readily hydrolyzed and comprises only 8-18% of adducts [8, 17]. The O6 position of guanine only makes up 5% of lesions but is the most stable of the three and persists in the DNA in the absence of MGMT enzyme activity [8, 17]. O6-methylguanine is considered to be the most lethal of the alkyl

*O-6-Methylguanine-DNA Methyltransferase*. The MGMT enzyme reduces the cytotoxicity of O6 alkylating agents like TMZ by catalyzing the transfer of methyl groups from the O6 position of guanine to a cysteine residue within the active site of the MGMT enzyme [1]. The transfer of ethyl or alkyl groups to MGMT renders the MGMT enzyme inactive, leading to its degra‐ dation via the proteosomal pathway [18]. Therefore, continued MGMT function requires the de novo expression of MGMT protein in order to provide resistance to chemotherapeutic agents. Unfortunately, many GBM tumors exhibit increased MGMT expression, which reduces the effectiveness of alkylating agents such as TMZ. GBM tumors with hypermethylated MGMT

adducts. For a visual representation of these common adducts, see Figure 1.

to continue despite the damage.

336 Advances in DNA Repair

supplemented with findings from our laboratory.

**Figure 1.** Common temozolomide-induced DNA lesions appear on guanine and adenine. N7-methylguanine, N3 methyladenine and O6-methylguanine DNA adducts account for roughly 70%, 10% and 5% of these lesions respective‐ ly. O6-methylguanine adducts are the most cytotoxic, yet are readily repaired by MGMT, resulting in TMZ resistance. The less toxic N7-methylguanine and N3-methyladenine adducts are readily repaired by the base excision repair (BER) system. The inhibition of PARP blocks BER and increases toxicity.

*Poly (ADP-ribose) Polymerase 1*. PARP-1 is a cell-survival factor that functions in single-stranded break repair (SSBR) to maintain genomic integrity [19]. The zinc-finger domain of PARP-1 binds to DNA nicks and adds the polyanion ADP-ribose (PAR) to histone proteins H1 and H2B [20]. PAR addition to histones relaxes the 30nm chromatin allowing access of DNA repair enzymes to the DNA. Inhibition of PARP-1 in proliferating cells sensitizes cells to DNA damage resulting in cell cycle arrest [21].

## **2. O-6-methylguanine-DNA methyltransferase**

*MGMT Mechanism of Action*. The MGMT gene is located on chromosome band 10q26.3 spanning 300kb, with 5 exons and 4 introns. It encodes the 207 amino acid MGMT repair enzyme that is highly conserved among species and plays a critical role in maintaining the integrity of genomic DNA. The MGMT 1.2kb promoter region is TATA-and CAAT-box free with numerous CpG islands. Expression levels of MGMT vary significantly between tissues and can be regulated by glucocorticoids, cAMP, protein kinase C, and DNA damage [22].

The transcription factor Sp1 functions in transcriptional regulation of the MGMT gene, and CpG methylation within the promoter sequence affects chromatin structure to affect Sp1 access to the promoter site [23]. Methylation of specific CpG clusters in the promoter is correlated with MGMT gene silencing [24]. However, the overall amount, location, and homogeneity of MGMT promoter methylation is variable in GBM [25]. Dunn et al. investigated 109 newly diagnosed GBMs, and found that 58 tumors had an elevated methylation status compared to non-neoplastic brain tissue (≥ 9% methylated). Furthermore, 19 of the tumors examined with a methylation status greater than 35% correlated with the highest 2-year survival rates [26]. It is not fully understood what determines MGMT promoter methylation levels, but recent evidence indicates that p53 may play a role. Using human lung cancer cells, Lai et al. showed that the knockdown of p53 increases MGMT promoter methylation in wild type p53 lung cancer cells [27] while Srivenugopal et al. reported that inducible p53 expression suppresses MGMT levels in a p53-null lung cancer cell line [28]. In contrast, published work by Wang et al. suggests that hypermethylation of CpG islands within the MGMT gene does not strictly correlate with reduced MGMT protein expression [29]. A number of studies have highlighted the variability in MGMT promoter methylation and MGMT gene expression levels indicating both a variability in MGMT activity within and between tumors [30]. More recently, Kanemoto et al. performed deep sequencing analyses of the entire MGMT promoter to develop a diagnostic assay for progression-free survival of GBM patients based upon hypermethylation of CpG islands. Despite the evidence that variability in MGMT promoter methylation does exist, the data confirm the general hypothesis that hypermethylation of the MGMT promoter does correlate with reduced MGMT enzyme activity [31].

The MGMT enzyme functions as both a transferase and acceptor of alkyl-groups. MGMT activity does not require cofactors or other enzymes, rapidly removes DNA adducts from the O6 position of guanine, and transfers them to an internal cysteine residue (Cys145) within the enzyme active site [32]. This reaction is stoichiometric and once the MGMT protein has been alkylated, it is inactivated and undergoes ubiquitin-mediated degradation [33].

Most alkylating agents used to therapeutically induce cell death target the O6-methylguanine adduct. While MGMT primarily repairs O6-methylguanine DNA adducts, it has the ability to repair adducts of greater size (i.e. O6-ethylguanine) as well as the minor alkylation product O4-methylthymine. MGMT-mediated repair pathways correct the damage caused by alkylat‐ ing chemotherapeutic agents utilized in the treatment of gliomas, melanomas, carcinoid tumors, and lymphomas, such as carmustine, temozolomide, streptozotocin, procarbazine, and dacarbazine [32].

*Poly (ADP-ribose) Polymerase 1*. PARP-1 is a cell-survival factor that functions in single-stranded break repair (SSBR) to maintain genomic integrity [19]. The zinc-finger domain of PARP-1 binds to DNA nicks and adds the polyanion ADP-ribose (PAR) to histone proteins H1 and H2B [20]. PAR addition to histones relaxes the 30nm chromatin allowing access of DNA repair enzymes to the DNA. Inhibition of PARP-1 in proliferating cells sensitizes cells to DNA

*MGMT Mechanism of Action*. The MGMT gene is located on chromosome band 10q26.3 spanning 300kb, with 5 exons and 4 introns. It encodes the 207 amino acid MGMT repair enzyme that is highly conserved among species and plays a critical role in maintaining the integrity of genomic DNA. The MGMT 1.2kb promoter region is TATA-and CAAT-box free with numerous CpG islands. Expression levels of MGMT vary significantly between tissues and can be regulated by glucocorticoids, cAMP, protein kinase C, and DNA damage [22]. The transcription factor Sp1 functions in transcriptional regulation of the MGMT gene, and CpG methylation within the promoter sequence affects chromatin structure to affect Sp1 access to the promoter site [23]. Methylation of specific CpG clusters in the promoter is correlated with MGMT gene silencing [24]. However, the overall amount, location, and homogeneity of MGMT promoter methylation is variable in GBM [25]. Dunn et al. investigated 109 newly diagnosed GBMs, and found that 58 tumors had an elevated methylation status compared to non-neoplastic brain tissue (≥ 9% methylated). Furthermore, 19 of the tumors examined with a methylation status greater than 35% correlated with the highest 2-year survival rates [26]. It is not fully understood what determines MGMT promoter methylation levels, but recent evidence indicates that p53 may play a role. Using human lung cancer cells, Lai et al. showed that the knockdown of p53 increases MGMT promoter methylation in wild type p53 lung cancer cells [27] while Srivenugopal et al. reported that inducible p53 expression suppresses MGMT levels in a p53-null lung cancer cell line [28]. In contrast, published work by Wang et al. suggests that hypermethylation of CpG islands within the MGMT gene does not strictly correlate with reduced MGMT protein expression [29]. A number of studies have highlighted the variability in MGMT promoter methylation and MGMT gene expression levels indicating both a variability in MGMT activity within and between tumors [30]. More recently, Kanemoto et al. performed deep sequencing analyses of the entire MGMT promoter to develop a diagnostic assay for progression-free survival of GBM patients based upon hypermethylation of CpG islands. Despite the evidence that variability in MGMT promoter methylation does exist, the data confirm the general hypothesis that hypermethylation of the MGMT promoter

The MGMT enzyme functions as both a transferase and acceptor of alkyl-groups. MGMT activity does not require cofactors or other enzymes, rapidly removes DNA adducts from the O6 position of guanine, and transfers them to an internal cysteine residue (Cys145) within the enzyme active site [32]. This reaction is stoichiometric and once the MGMT protein has been

alkylated, it is inactivated and undergoes ubiquitin-mediated degradation [33].

damage resulting in cell cycle arrest [21].

338 Advances in DNA Repair

**2. O-6-methylguanine-DNA methyltransferase**

does correlate with reduced MGMT enzyme activity [31].

*Clinical Implications of MGMT*. As noted above, MGMT specifically reverses the DNA damaging effects of TMZ, and hypermethylation of the promoter for the MGMT gene correlates with reduced MGMT production. This finding has clinical implications, as highlighted by Stupp et al. and Hegi et al. in companion 2005 papers [2, 34]. Stupp et al. reported results from a multicenter, randomized trial comparing adjuvant radiation therapy to radiation therapy plus temozolomide for the treatment of glioblastoma. The median survival for the temozolomide group was 14.6, versus 12.1 months in the radiation only group. The two year survival rate was 26.5% in the temozolomide group versus 10.4% in the radiation only group. Perhaps the more interesting findings came from subgroup analysis. Hegi et al. stratified the two treatment groups based on MGMT promoter methylation. Though less than half of the patients enrolled in the clinical trial had usable DNA methylation data, the results, summarized in Table 1, are nonetheless compelling. In nearly a decade since the publications of these papers, the conclu‐ sion that MGMT promoter methylation sensitizes malignant glioma to temozolomide has been confirmed in multiple clinical and cohort studies [35-38]. MGMT promoter methylation status is an important prognostic biomarker and it appears that MGMT methylation status should be considered when formulating the treatment plan [39].

## **3. Inhibition of O-6-methylguanine-DNA methyltransferase**

The key mechanism of resistance to alkylating agents in GBM is the presence of MGMT enzyme, and most human tumors exhibit high levels of MGMT expression and activity. As mentioned previously, elevated expression of MGMT is inversely correlated with survival [34, 40, 41]. Thus, suppression of MGMT activity could render cells more sensitive to alkylating agents, augmenting cytotoxicity.

*O6-benzylguanine*. Of the agents to target MGMT suppression, O6-benzylguanine (O6BG) was the first developed and was thought to have the greatest potential. O6BG is an MGMT substrate that inactivates MGMT in a suicide manner by binding to the protein. In the early/mid 1990's, studies using O6BG to suppress MGMT in GBM, both *in vitro* [42, 43] and *in vivo* [44], showed increased sensitivity and cytotoxicity to alkylating agents. These data initiated several clinical trials [45-49]. Unfortunately, patients in these early phase trials also exhibited significant hematological toxicity to O6BG and late phase trials were not pursued. Although O6BG may someday find a very useful place in neuro-oncology, the data from these clinical trials suggested that safer and more effective therapeutic approaches were needed to target MGMT.

**Table 1.** Data summarized from Hegi et al. 2005.

*Gene Therapy*. While O6BG was proving to be too toxic for systemic delivery, a promising method for treating malignant tumors was being developed, gene therapy. Several studies have targeted oncogenes both *in vitro* and *in vivo* using antisense oligonucleotides with varying success, as described in the review by Caffo et al. [50]. Further, multiple studies have applied RNA interference strategies to treatment of gliomas - a Pubmed search in September of 2014 using the key words "RNA interference and glioma" generated 707 citations. The disruption of genes via synthetic nucleotide sequences may provide a specific inhibition of tumor growth with minimal off-target effects.

In 2008, our lab used RNA interference *in vitro* to silence the MGMT gene in GBM cell lines expressing high (U138MG), low (U87MG) and non-detectable levels (LN229) of MGMT [51]. Baseline TMZ dose response curves indicated that GBM cells with high levels of MGMT expression exhibit the greatest resistance. The half maximum effective concentration (EC50) of TMZ was consistently higher in cells with higher MGMT expression, this finding was similar to previous reports [52]. After MGMT mRNA knockdown, U138 cells became more sensitive to TMZ as expected (Figure 2). Interestingly, we were unable to detect a gene expression change in the U87MG cells, even though siRNA treatment rendered these cells more sensitive to TMZ treatment. No gene knockdown occurred in the LN229 cells (MGMT was not detectable at baseline) and the EC50 in these cells did not change after attempting RNA interference. These data are consistent with the work of others [13, 53] suggesting that siRNA could be an effective therapeutic agent.

Although it is possible for siRNA to function efficiently using cells grown in culture, siRNA application within organisms is difficult. These siRNA macromolecules do not cross cell membranes easily and would make crossing the blood brain barrier problematic. Thus, novel delivery systems for siRNA are being investigated. One successful nanoparticle system being developed is the LipoTrust EX Oligo liposome delivery system [13]. Kato et al. reported that liposome delivery of siRNA to downregulate MGMT was effective in sensitizing GBM to TMZ in both *in vitro* and *in vivo* models [13]. Nanoparticles, whole cells and viruses, alone and in combination, are options being explored for delivery of synthetic nucleotides [54, 55]. It appears that gene therapy is a realistic possibility and may provide patients with "personalized medicine" in the near future. Yet, there are major concerns with the use of the technology, including off-target gene effects. Further, there are concerns about the use of viruses for gene therapy because of their potential to induce mutations. Oncolytic viruses, which preferentially lyse tumor cells, can become more selective and effective when engineered to possess tumor specific transgenes. The conditionally replicating adenovirus is one such oncolytic virus that not only targets the tumor cell but also possesses a gene that blocks the recruitment of the transcriptional coactivator p300 to the MGMT promoter [56].

**Methylation Status Radiation Alone Radiation and Temozolomide**

*Gene Therapy*. While O6BG was proving to be too toxic for systemic delivery, a promising method for treating malignant tumors was being developed, gene therapy. Several studies have targeted oncogenes both *in vitro* and *in vivo* using antisense oligonucleotides with varying success, as described in the review by Caffo et al. [50]. Further, multiple studies have applied RNA interference strategies to treatment of gliomas - a Pubmed search in September of 2014 using the key words "RNA interference and glioma" generated 707 citations. The disruption of genes via synthetic nucleotide sequences may provide a specific inhibition of tumor growth

In 2008, our lab used RNA interference *in vitro* to silence the MGMT gene in GBM cell lines expressing high (U138MG), low (U87MG) and non-detectable levels (LN229) of MGMT [51]. Baseline TMZ dose response curves indicated that GBM cells with high levels of MGMT expression exhibit the greatest resistance. The half maximum effective concentration (EC50) of TMZ was consistently higher in cells with higher MGMT expression, this finding was similar to previous reports [52]. After MGMT mRNA knockdown, U138 cells became more sensitive to TMZ as expected (Figure 2). Interestingly, we were unable to detect a gene expression change in the U87MG cells, even though siRNA treatment rendered these cells more sensitive to TMZ treatment. No gene knockdown occurred in the LN229 cells (MGMT was not detectable at baseline) and the EC50 in these cells did not change after attempting RNA interference. These data are consistent with the work of others [13, 53] suggesting that siRNA could be an effective

Although it is possible for siRNA to function efficiently using cells grown in culture, siRNA application within organisms is difficult. These siRNA macromolecules do not cross cell membranes easily and would make crossing the blood brain barrier problematic. Thus, novel delivery systems for siRNA are being investigated. One successful nanoparticle system being developed is the LipoTrust EX Oligo liposome delivery system [13]. Kato et al. reported that liposome delivery of siRNA to downregulate MGMT was effective in sensitizing GBM to TMZ

Patients 46 46 Median progression free survival (months) 5.9 10.3 Median overall survival (months) 15.3 21.7 2 year survival (%) 22.7 46

Patients 54 60 Median progression free survival (months) 4.4 5.3 Median overall survival (months) 11.8 12.7 2 year survival (%) 0 13.8

**MGMT Methylated**

340 Advances in DNA Repair

**MGMT Unmethylated**

**Table 1.** Data summarized from Hegi et al. 2005.

with minimal off-target effects.

therapeutic agent.

*p53.* DNA damage from alkylating agents, ionizing radiation and ultraviolet light induces p53 activity. The role p53 plays in MGMT expression is not fully understood. Knockdown of p53 increases MGMT promoter methylation [27], yet overexpression of wild-type p53 suppresses MGMT and renders human cancer cells more sensitive to TMZ [28]. A previous study also reported MGMT downregulation in p53-null osteosarcoma cells with the introduction of wildtype p53 [57]. Grombacher et al. demonstrated that the overexpression of p53 reduced basal MGMT promoter activity in rodent cells [58]. The mechanism of p53-mediated MGMT knockdown may be through physical binding of the MGMT promoter without altering the methylation status [59]. It has also been shown that p53 prevents Sp1 from binding to the promoter [60]. Because overexpression of p53 may suppress MGMT, targeting p53 may render GBM more sensitive to TMZ. Levetiracetam is often used in GBM patients to manage seizures, but its use may further benefit patients by augmenting p53-mediated MGMT suppression [61]. Interferon beta (IFN-β) was shown to mediate cytotoxicity in human GBM cell lines [62], most likely because IFN-β induces p53 and improves the response of GBM cells to TMZ treatment [63, 64]. More recently, the use of a mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor has shown promise as a TMZ adjuvant by activating p53 [65]. Molecules targeting miRNAs may also be effective at p53-mediated suppression of MGMT. For example, ways to suppress miR-21 may also reduce MGMT levels via p53 activation [66]. The utility of targeting p53 may be limited [64] due to the high incidence of p53 mutation in GBM [63].

*Other MGMT Inhibitors*. Other targets have been suggested for regulation of MGMT levels in the cell. For example, valproic acid has become an established anti-cancer drug because of it's role in histone deacetylation, but it has also been shown to downregulate MGMT in GBM cells [67]. The proteasome inhibitors Bortezomib and MG132 also suppress MGMT transcription [68, 69]. Altering the expression of tumor suppressor genes may also be an effective treatment strategy. For example, miR-181d has been shown to down regulate MGMT possibly via the Kras-related Pi3K/AKT and MapK/ERK pathways [70]. Lastly, the cytokine interleukin-24 downregulates MGMT expression in human melanoma cells and the DNA crosslinking agent cisplatin suppresses MGMT in leukemia cells [71, 72].

It is worth noting that individualized therapy based solely on MGMT promoter methylation alone may not always be advantageous. MGMT promoter methylation may not correlate with TMZ sensitivity and survival in some populations [73]. Although it has been reported that the level of MGMT mRNA and protein expression is correlated with promoter methylation status

**Figure 2.** U138MG (high MGMT expression), U87MG (low MGMT expression) and LN229 (no MGMT detected) dose response curves from siRNA experiments (March 2008). The difference in EC50 between the siRNA treated group and the negative and untreated control group was statistically different for U138MG (P<0.0001) and U87MG (P<0.01), with no difference in the LN229 cell line. The figure legend represents the EC50 values for each treatment group in µM of TMZ.

[74-76], this correlation does not hold true in all cases [73, 76-80]. Reports of GBMs with unmethylated MGMT promoter regions and low MGMT mRNA expression as well as GBMs with methylated MGMT promoter regions expressing high MGMT mRNA levels suggest that methylation-independent pathways may alter MGMT mRNA levels [74, 76, 77, 81]. Evidence suggests that mechanisms of post-transcriptional regulation alter MGMT protein expression since protein analysis of MGMT does not correlate with mRNA [76]. Recent data suggest that miRNA regulation of MGMT may explain these discrepancies and miRNAs are currently being investigated as therapeutic targets [70, 82, 83]. Because MGMT expression appears be predic‐ tive of progression free and overall survival [74], adequate assessment of tumors may need to include MGMT mRNA and/or protein expression.

*MGMT Summary*. MGMT plays a critical role in maintaining the integrity of genomic DNA. Unfortunately, elevated MGMT expression is correlated with poorer prognosis in cancer patients. Tumors expressing MGMT will not respond well to alkylating chemotherapy because MGMT corrects O6-methylguanine. Screening the tumor tissue not only for MGMT (expres‐ sion and methylation status), but also for other genes that may suppress or increase MGMT expression will be important for successful management of GBM. Clinicians should be guarded, however, when determining the treatment strategy because not all GBMs display the same magnitude, locations and homogeneity of methylations [25]. Although many therapeutic targets have been found to suppress MGMT activity, research is still needed to determine which of these molecules will effectively suppress MGMT while being safely administered to the patient.

## **4. Poly (ADP-ribose) polymerase 1**

**Figure 2.** U138MG (high MGMT expression), U87MG (low MGMT expression) and LN229 (no MGMT detected) dose response curves from siRNA experiments (March 2008). The difference in EC50 between the siRNA treated group and the negative and untreated control group was statistically different for U138MG (P<0.0001) and U87MG (P<0.01), with no difference in the LN229 cell line. The figure legend represents the EC50 values for each treatment group in µM of

TMZ.

342 Advances in DNA Repair

*PARP-1 Mechanism of Action*. The human Poly (ADP-ribose) Polymerase (PARP) gene family is evolutionarily conserved and codes for 17 different enzymes. The most important member of the PARP family, PARP-1, is a key DNA repair enzyme located on chromosome 1q42 and is responsible for the majority of PARP activity in the cell. As a component of the base excision repair (BER) pathway, PARP-1 binds to single-strand DNA breaks, catalyzes the formation of ADP-ribose polyanions from its substrate nicotinamide adenine dinucleotide, and recruits additional repair enzymes to the damaged strand. In addition to its central role in DNA repair, the PARP-1 enzyme also regulates other vital biological functions as reviewed in Krishnaku‐ mar and Kraus, 2010 [84]. While PARP-1 protein expression is typically low in normal brain tissue, it is highly expressed in GBMs [85]. The transcription of PARP-1 appears to be primarily regulated by the transcription factors SP1 and NFI, but, AP2, YY1 and ETS also bind to promoter sites of the PARP-1 gene [86].

*Clinical Implications of PARP-1*. In GBM, the effectiveness of radiation and chemotherapy is mitigated by normal cellular DNA repair mechanisms. It follows that interfering with DNA repair will enhance DNA damaging treatments. A number of preclinical studies confirm that PARP inhibition strengthens the efficacy of several DNA damaging anticancer therapies including radiation, DNA methylating agents, and topoisomerase I inhibitors [87]. A particu‐ larly exciting application of PARP inhibition is in cancer cells with defects in homologous recombination. The loss of both single strand repair and repair of double strand DNA breaks is fatal [88]. This example of synthetic lethality demonstrates that these two loss-of-function mutations prove fatal, whereas either mutation alone is not. Clinical trials have utilized PARP inhibitors in two ways: in combination with DNA damaging therapies and as a single agent for tumors deficient in homologous repair (e.g., BRCA1/2 mutated breast cancers) [87]. Further, epidermal growth factor receptor (EGFR) status may act as a predictive biomarker for PARP inhibition sensitivity [89]. The EGFR gene is overexpressed in ~50% of GBMs [90]. Nearly 20% of grade III and IV tumors possess the constitutively active class III variant (EGFRvIII) and correlates with elevated expression of the gene [91]. Clark Chen's group recently determined that GBM cells over-expressing EGFRvIII were dependent on the BER system and PARP-1 related function for cell survival [89]. Following pharmacological inhibition or PARP-1 silencing with siRNAs, cytotoxicity was increased in GBM cells expressing elevated levels of EGFRvIII. It was suggested the increased cytoxicity was due to the inability to correct the damage caused by reactive oxygen species and the effect was greater when coupled with radiation [89].

## **5. Inhibition of poly (ADP-ribose) polymerase 1**

Cancer treatments utilizing ionizing radiation and DNA alkylating agents damage DNA, which if not repaired causes cell death. Inhibition of PARP-1 contributes to the sensitization of tumor cells to these treatments and is the basis for multiple preclinical and clinical studies with PARP inhibitors in combination with classical therapies [92].

*Benzamides*. The inhibition of PARP-1 and the role of PARP-1 inhibition has been of interest since the early 1980's when it was demonstrated that PARP could be inhibited using nicoti‐ namide analogues [93]. The initial interest in PARP inhibition was to determine the role of the enzyme in the cell. The ability to inhibit PARP was responsible for elucidation of the function of PARP and its role in DNA repair. Using 3-aminobenzamide, Sidney Shall's group demon‐ strated that inhibition of PARP resulted in disruption of the repair of DNA breaks created by treatment with the DNA alkylating agent, dimethyl sulfate. Additionally, they reported that PARP inhibition enhanced the efficacy of dimethyl sulfate as a cytotoxic agent [94]. This study was the first to suggest that combined treatments with DNA alkylating agents and PARP inhibition could be effective in the treatment of cancer. The benzamides were essential in determining the role of PARP and providing "proof of principle" that PARP inhibition could play a role in cancer treatment and increase the efficiency of DNA damaging agents. However, the benzamides are relatively weak PARP inhibitors and have been shown to interfere with cellular pathways not associated with PARP [95]. More recently, specific PARP inhibitors have been produced and several are currently in clinical trials. Second and third generation PARP inhibitors are more potent and require markedly lower effective concentrations to reduce 50% of PARP's activity [96].

*New Generation Inhibitors*. PARP inhibition results from two different but complementary mechanisms, blocking PARP catalytic activity and the release of the enzyme from the DNA. The enzyme needs to be poly(ADP-ribosyl)ated to dissociate from DNA and if that ability is blocked by the catalytic inhibitor, it remains bound causing a physical obstruction to the repair of the DNA break. This was first proposed by Masahiko Satoh and Tomas Lindahl in 1992, who demonstrated that the repair of nicked plasmid DNA by nuclear extracts was not dependent on PARP, indicating that other repair pathways were still effective [97]. However, if PARP was present then its substrate, NAD+, was necessary for repair to occur. If this PARP-NAD+ repair requirement was blocked by 3-aminobenzamide, which com‐ petes for the NAD+ binding site, DNA repair was stopped. This indicated that inactive PARP impeded DNA repair, not only did PARP not recruit repair enzymes to the site of damage but also physically blocked other DNA repair pathways. Recent work expanded our understanding of the role of PARP inhibitors in DNA binding. DNA damage pro‐ duced by alkylating agents administered concomitantly with a PARP inhibitor resulted in increased PARP–DNA binding compared to PARP–DNA binding with either the alkylat‐ ing agent or PARP inhibitor alone [98]. In a similar study it was demonstrated that effectiveness of a PARP inhibitor might not only be its ability to inhibit the catalytic ability of the enzyme but also its ability to trap the enzyme on the DNA strand [99]. Using three potent PARP inhibitors (Olaparib (AZD-2281), veliparib (ABT-888), and niraparib (MK-4827)), currently being investigated clinically, it was demonstrated that all are effective in suppression of catalytic activity, having IC50 values that are in the low nanomolar range [100]. If we were to assume that the function of PARP inhibitors is solely explained by catalytic inhibition these three drugs should have similar effect and that effect should not differ from PARP deletion or silencing. However, it was reported that following treat‐ ment with the DNA alkylating agent, MMS, that while all three PARP inhibitors were effective in inhibiting catalytic activity, these clinically relevant PARP inhibitors differ markedly in their potency to induce cytotoxic PARP-DNA complexes. The authors indicated that, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug and suggested that further PARP inhibitor studies should examine both aspects, catalytic inhibition and DNA trapping, in inhibitor evaluation [100].

larly exciting application of PARP inhibition is in cancer cells with defects in homologous recombination. The loss of both single strand repair and repair of double strand DNA breaks is fatal [88]. This example of synthetic lethality demonstrates that these two loss-of-function mutations prove fatal, whereas either mutation alone is not. Clinical trials have utilized PARP inhibitors in two ways: in combination with DNA damaging therapies and as a single agent for tumors deficient in homologous repair (e.g., BRCA1/2 mutated breast cancers) [87]. Further, epidermal growth factor receptor (EGFR) status may act as a predictive biomarker for PARP inhibition sensitivity [89]. The EGFR gene is overexpressed in ~50% of GBMs [90]. Nearly 20% of grade III and IV tumors possess the constitutively active class III variant (EGFRvIII) and correlates with elevated expression of the gene [91]. Clark Chen's group recently determined that GBM cells over-expressing EGFRvIII were dependent on the BER system and PARP-1 related function for cell survival [89]. Following pharmacological inhibition or PARP-1 silencing with siRNAs, cytotoxicity was increased in GBM cells expressing elevated levels of EGFRvIII. It was suggested the increased cytoxicity was due to the inability to correct the damage caused by reactive oxygen species and the effect was greater when coupled with

Cancer treatments utilizing ionizing radiation and DNA alkylating agents damage DNA, which if not repaired causes cell death. Inhibition of PARP-1 contributes to the sensitization of tumor cells to these treatments and is the basis for multiple preclinical and clinical studies

*Benzamides*. The inhibition of PARP-1 and the role of PARP-1 inhibition has been of interest since the early 1980's when it was demonstrated that PARP could be inhibited using nicoti‐ namide analogues [93]. The initial interest in PARP inhibition was to determine the role of the enzyme in the cell. The ability to inhibit PARP was responsible for elucidation of the function of PARP and its role in DNA repair. Using 3-aminobenzamide, Sidney Shall's group demon‐ strated that inhibition of PARP resulted in disruption of the repair of DNA breaks created by treatment with the DNA alkylating agent, dimethyl sulfate. Additionally, they reported that PARP inhibition enhanced the efficacy of dimethyl sulfate as a cytotoxic agent [94]. This study was the first to suggest that combined treatments with DNA alkylating agents and PARP inhibition could be effective in the treatment of cancer. The benzamides were essential in determining the role of PARP and providing "proof of principle" that PARP inhibition could play a role in cancer treatment and increase the efficiency of DNA damaging agents. However, the benzamides are relatively weak PARP inhibitors and have been shown to interfere with cellular pathways not associated with PARP [95]. More recently, specific PARP inhibitors have been produced and several are currently in clinical trials. Second and third generation PARP inhibitors are more potent and require markedly lower effective concentrations to reduce 50%

radiation [89].

344 Advances in DNA Repair

of PARP's activity [96].

**5. Inhibition of poly (ADP-ribose) polymerase 1**

with PARP inhibitors in combination with classical therapies [92].

*Inhibitors Versus Deletion or Silencing*. It is becoming clear that there are subtle, but important differences between PARP deletion versus PARP inhibition. PARP-1 knockout mice are viable and fertile as are PARP-2 knockout mice. However, the deletion of both enzymes is lethal [101]. This indicates that while either enzyme can fill the DNA repair role, that role is essential. Additionally, it has been shown that PARP-1-/-knockout mice are more sensitive to radiation than wildtype mice [102]. In addition to knockout mice, studies examining the effect of PARP silencing using RNA interference have been effective in reduction of PARP and PARP activity and have increased radiosensitivity in a manner equivalent to PARP-1-/-knockout mice [103]. In our hands, silencing of PARP-1 in glioblastoma cell lines was generally not as effective as treatment with a PARP inhibitor [104]. The greater efficacy of PARP inhibitors compared to PARP knockout or PARP silencing likely has to do with the mode of action of the inhibitors. This is an important consideration as it has been shown that inhibitors act on both PARP-1 and PARP-2, usually with similar potency and thus inhibition is more similar to the deletion of both enzymes. Additionally, it is well established that PARP in combination with an inhibitor blocks DNA repair in at least two ways.

*PARP and Glioblastoma*. Temozolomide acts by specific methylation of the DNA bases guanine and adenine resulting in inappropriate pairing during DNA replication. O6-methylguanine will trigger the DNA mismatch repair (MMR) pathway and become highly cytotoxic unless it is corrected by MGMT. However, nearly 80% of the total methylation events resulting from TMZ treatment are the N7-methylguanine and N3-methyladenine that trigger the BER system. The disruption of the BER pathway through PARP inhibition renders these lesions cytotoxic and helps overcome the MGMT related TMZ resistance. Another important consideration is the use of PARP inhibitors to increase TMZ sensitivity in tumors that exhibit MMR deficiencies, a relatively common occurrence in sporadic cancers. In these tumors, the lack of MMR, causes the cell to overlook the O6-methylguanine lesion rendering TMZ ineffective. Treatment with a PARP inhibitor renders the N3-methyladenine and N7-methylguanine lesions cytotoxic [105]. However, it has been reported that MMR deficiency does not seem to be responsible for mediating TMZ resistance in adult GBM [106].

*Targeting PARP-1 and MGMT*. In 2011 we examined the role that PARP-1 inhibition plays in altering GBM cell lines' response to TMZ [104]. We examined any changes in TMZ effectiveness in the GBM cells using either the PARP inhibitor, 3-aminobenzamide (3-AB) and compared its effect to silencing of PARP-1 using RNA interference. In our hands, the use of the PARP inhibitor was generally as, or more, effective than PARP silencing. Additionally, we examined the response to TMZ following PARP-1 silencing combined with MGMT silencing by RNA interference. The response to TMZ following silencing of both PARP-1 and MGMT was compared to the response to TMZ following PARP inhibition and MGMT silencing. In three of four GBM cell lines, the PARP inhibitor, 3-AB, combined with MGMT silencing rendered cells more sensitive to TMZ compared to silencing of both PARP-1 and MGMT or TMZ alone. These data suggest that GBM cells may be more sensitive to PARP inhibition versus PARP silencing, but also suggest that targeting both MGMT and the BER system may result in lasting TMZ-induced lesions leading to cell death.

*PARP-1 Summary*. PARP-1 and PARP-2 play critical roles which have been demonstrated by the lethality seen when both genes are deleted. One well defined role for these en‐ zymes is DNA repair via the base excision repair pathway. Unfortunately, many cancer treatments including radiation and many types of chemotherapeutic agents act by damag‐ ing DNA. Much of this damage can be repaired through the actions of PARP. PARP inhibitors have been shown to bind effectively to both PARP-1 and PARP-2, block the enzymatic activity and may trap the inactivated PARP on the DNA lesion, effectively blocking additional DNA repair mechanisms. These actions have been shown to increase

the sensitivity to radiation and DNA alkylating chemotherapies in preclinical studies. However, while promising, the result of most clinical trials has been somewhat disappoint‐ ing as the PARP inhibitors as chemopotentiating agents has been limited by an increase in toxicities, necessitating dose reductions of the cytotoxic chemotherapeutic agent and the PARP inhibitor. Ongoing research re-examining the mechanism of action of the PARP inhibitors, including those that bind in sites other than the PARP catalytic site, may eliminate some of the off target binding and reduce inhibition of other members of the PARP family. Also, continued efforts to determine other pathways affected by the PARP inhibitors are necessary to make adequate decisions about clinical usage.

## **6. Conclusions**

treatment with a PARP inhibitor [104]. The greater efficacy of PARP inhibitors compared to PARP knockout or PARP silencing likely has to do with the mode of action of the inhibitors. This is an important consideration as it has been shown that inhibitors act on both PARP-1 and PARP-2, usually with similar potency and thus inhibition is more similar to the deletion of both enzymes. Additionally, it is well established that PARP in combination with an inhibitor

*PARP and Glioblastoma*. Temozolomide acts by specific methylation of the DNA bases guanine and adenine resulting in inappropriate pairing during DNA replication. O6-methylguanine will trigger the DNA mismatch repair (MMR) pathway and become highly cytotoxic unless it is corrected by MGMT. However, nearly 80% of the total methylation events resulting from TMZ treatment are the N7-methylguanine and N3-methyladenine that trigger the BER system. The disruption of the BER pathway through PARP inhibition renders these lesions cytotoxic and helps overcome the MGMT related TMZ resistance. Another important consideration is the use of PARP inhibitors to increase TMZ sensitivity in tumors that exhibit MMR deficiencies, a relatively common occurrence in sporadic cancers. In these tumors, the lack of MMR, causes the cell to overlook the O6-methylguanine lesion rendering TMZ ineffective. Treatment with a PARP inhibitor renders the N3-methyladenine and N7-methylguanine lesions cytotoxic [105]. However, it has been reported that MMR deficiency does not seem to be responsible for

*Targeting PARP-1 and MGMT*. In 2011 we examined the role that PARP-1 inhibition plays in altering GBM cell lines' response to TMZ [104]. We examined any changes in TMZ effectiveness in the GBM cells using either the PARP inhibitor, 3-aminobenzamide (3-AB) and compared its effect to silencing of PARP-1 using RNA interference. In our hands, the use of the PARP inhibitor was generally as, or more, effective than PARP silencing. Additionally, we examined the response to TMZ following PARP-1 silencing combined with MGMT silencing by RNA interference. The response to TMZ following silencing of both PARP-1 and MGMT was compared to the response to TMZ following PARP inhibition and MGMT silencing. In three of four GBM cell lines, the PARP inhibitor, 3-AB, combined with MGMT silencing rendered cells more sensitive to TMZ compared to silencing of both PARP-1 and MGMT or TMZ alone. These data suggest that GBM cells may be more sensitive to PARP inhibition versus PARP silencing, but also suggest that targeting both MGMT and the BER system may result in lasting

*PARP-1 Summary*. PARP-1 and PARP-2 play critical roles which have been demonstrated by the lethality seen when both genes are deleted. One well defined role for these en‐ zymes is DNA repair via the base excision repair pathway. Unfortunately, many cancer treatments including radiation and many types of chemotherapeutic agents act by damag‐ ing DNA. Much of this damage can be repaired through the actions of PARP. PARP inhibitors have been shown to bind effectively to both PARP-1 and PARP-2, block the enzymatic activity and may trap the inactivated PARP on the DNA lesion, effectively blocking additional DNA repair mechanisms. These actions have been shown to increase

blocks DNA repair in at least two ways.

346 Advances in DNA Repair

mediating TMZ resistance in adult GBM [106].

TMZ-induced lesions leading to cell death.

Surgical resection, radiation and use of TMZ is currently the standard of care for GBM patients. The alkylating agent, TMZ, induces lesions at the N7 and O6 positions of guanine and N3 position of adenine. However, many of these tumors express MGMT which promptly corrects the most cytotoxic lesion, the O6-methylguanine adduct. Tumors expressing MGMT are, therefore, inherently resistant to TMZ. MGMT inhibition improves the response to TMZ, but MGMT inhibition as standard therapy is still in development. O6BG, although too toxic to give systemically, may find itself useful for future therapy if delivery to the central nervous system can be improved. Gene therapy, with the enhancement of the delivery of synthetic nucleotides like the LipoTrust and oncolytic viruses, may become the standard of care in the future. There are several other molecules targeting pathways that influence MGMT and many more will surely emerge.

The majority (~80%) of these TMZ-induced DNA alkyl adducts, N7-methylguanine and N3 methyladenine, are repaired by the BER system. After the mismatched base has been removed, PARP-1 plays a role in repairing DNA breaks by binding and recruiting other BER proteins. Because of its role in the BER system, PARP inhibitors also improve the response to TMZ. While the results from numerous clinical trials have been disappointing due to systemic toxicities, new inhibitors may improve outcomes.

Modulation of TMZ resistance through the MGMT and BER pathways is clinically viable. New combinations of existing strategies may prove to further compliment TMZ and augment its effectiveness. Although several approaches have been used to modulate PARP and MGMT pathways, molecular screening should be used to identify targets with the greatest therapeutic potential. For example, pre-treatment assessment for MGMT and EGFR expression would provide information regarding the susceptibility to TMZ and PARP inhibitors, respectively. With the growing understanding of the pathways involved with DNA repair, the design of novel strategies or the use of combinations of existing thera‐ pies may improve GBM outcome.

## **Author details**

Johnathan E. Lawrence1 , Cathy E. Bammert1 , Robert J. Belton Jr.2 , Richard A. Rovin3 and Robert J. Winn1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Upper Michigan Brain Tumor Center and College of Arts and Sciences, Northern Michigan University, Marquette, MI, USA

2 Upper Michigan Brain Tumor Center and Biology Department, Northern Michigan University, Marquette, MI, USA

3 Upper Michigan Brain Tumor Center and Neurosurgery, Marquette General Hospital, Marquette, MI, USA

## **References**

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**Author details**

348 Advances in DNA Repair

Robert J. Winn1\*

Johnathan E. Lawrence1

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