hypoxIa and metabolIsm Hypoxia, DNA repair and genetic ... · [email protected]...

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Most solid human tumours contain regions of acute and chronic hypoxia or anoxia that can herald a nega- tive clinical prognosis for the cancer patient owing to local resistance and systemic metastases. Many studies of hypoxia-induced signalling responses have focused on the role of the transcription factor hypoxia inducible factor 1α (HIF1α) and angiogenesis. However, evidence also supports the concept that hypoxia can drive and maintain genetic instability and a mutator phenotype. Genetic instability can arise as a function of hypoxia- mediated resistance to apoptosis and decreased DNA repair, leading to increased rates of mutagenesis and altered chromatin biology. This might be particularly true in proliferating cells that have adapted to low O 2 levels and continue to proliferate in the context of com- promised DNA repair 1 . Herein, we focus on the effects of acute versus chronic hypoxia and hypoxic cellular adaptation on the ATM (ataxia telangiectasia mutated) and/or ATR (ataxia telangiectasia and Rad3-related) kinases, which mediate cell cycle checkpoint control; and the sensing and repair of DNA breaks. We review the data that supports the view of hypoxia as a driving force in genetic instability and the acquisition of the metastatic phenotype. Finally, we discuss the concept that hypoxia-associated genetic instability and faulty DNA repair might be a target for novel cancer therapies. Intra-tumoural hypoxia and tumour progression The tumour microenvironment is highly dynamic and contains subpopulations of cancer cells with dynamic gradients of cellular metabolism, O 2 content, pH, genomic stability and propensity for aggressive behaviours. For example, tumours can contain cells that have been differentially exposed to acute hypoxia (such as low intracellular O 2 levels for minutes to hours) and then reoxygenated. This can reoccur and lead to ‘cycling’ hypoxia. Cells could also be exposed to a more prolonged chronic hypoxia (low intracellu- lar O 2 levels for hours to days) before undergoing cell death or reoxygenation. A major concept emerging in clinical oncology is the detection of these tumour cell subpopulations and the determination of their effect both on prognosis and the response to current thera- pies such as surgery, radiotherapy, endocrine therapy or chemotherapy 2 . A dynamic tumour microenvironment can not only promote systemic metastasis, but also significantly modify drug action. However, preclinical studies of molecular-targeted agents that inhibit cancer cell signal- ling pathways are rarely conducted under varied micro- environmental conditions (such as within normoxic versus hypoxic conditions) to determine tumour cell kill under conditions that exist in solid tumours. This potential effect of the tumour microenvironment and intratumoural heterogeneity is important in the context of developing individualized cancer medicine and the judicious use of novel molecular-targeted agents. Transcription and translation in acute and chronic hypoxia. Intratumoural hypoxia is an adverse clinical prognostic factor that is associated with decreased disease- free survival for cancers involving the prostate, cervix, breast, musculoskeletal tissues and the head and neck 3–6 . Departments of Medical Biophysics and Radiation Oncology, University of Toronto and Ontario Cancer Institute and Princess Margaret Hospital (University Health Network), 610 University Avenue, Toronto, Ontario, M5G2M9, Canada. Correspondence to R.G.B. e-mail: [email protected] doi:10.1038/nrc2344 Published online 14 February 2008 Hypoxic cellular adaptation Cells can continue to proliferate in decreasing O 2 gradients by altering the transcription and translation of genes that are involved in angiogenesis and cell invasion, cell metabolism and cell survival. Hypoxia, DNA repair and genetic instability Robert G. Bristow and Richard P. Hill Abstract | Areas of hypoxic tumour tissue are known to be resistant to treatment and are associated with a poor clinical prognosis. There are several reasons why this might be, including the capacity of hypoxia to drive genomic instability and alter DNA damage repair pathways. Significantly, current models fail to distinguish between the complexities of the hypoxic microenvironment and the biological effects of acute hypoxia exposures versus longer-term, chronic hypoxia exposures on the transcription and translation of proteins involved in genetic stability and cell survival. Acute and chronic hypoxia might lead to different biology within the tumour and this might have a direct effect on the design of new therapies for the treatment of hypoxic tumours. HYPOXIA AND METABOLISM REVIEWS 180 | MARCH 2008 | VOLUME 8 www.nature.com/reviews/cancer © 2008 Nature Publishing Group

Transcript of hypoxIa and metabolIsm Hypoxia, DNA repair and genetic ... · [email protected]...

Page 1: hypoxIa and metabolIsm Hypoxia, DNA repair and genetic ... · rob.bristow@rmp.uhn.on.ca doi:10.1038/nrc2344 Published online 14 February 2008 Hypoxic cellular adaptation Cells can

Most solid human tumours contain regions of acute and chronic hypoxia or anoxia that can herald a nega-tive clinical prognosis for the cancer patient owing to local resistance and systemic metastases. Many studies of hypoxia-induced signalling responses have focused on the role of the transcription factor hypoxia inducible factor 1α (HIF1α) and angiogenesis. However, evidence also supports the concept that hypoxia can drive and maintain genetic instability and a mutator phenotype. Genetic instability can arise as a function of hypoxia-mediated resistance to apoptosis and decreased DNA repair, leading to increased rates of mutagenesis and altered chromatin biology. This might be particularly true in proliferating cells that have adapted to low O2 levels and continue to proliferate in the context of com-promised DNA repair1.

Herein, we focus on the effects of acute versus chronic hypoxia and hypoxic cellular adaptation on the ATM (ataxia telangiectasia mutated) and/or ATR (ataxia telangiectasia and Rad3-related) kinases, which mediate cell cycle checkpoint control; and the sensing and repair of DNA breaks. We review the data that supports the view of hypoxia as a driving force in genetic instability and the acquisition of the metastatic phenotype. Finally, we discuss the concept that hypoxia-associated genetic instability and faulty DNA repair might be a target for novel cancer therapies.

Intra-tumoural hypoxia and tumour progressionThe tumour microenvironment is highly dynamic and contains subpopulations of cancer cells with dynamic gradients of cellular metabolism, O2 content,

pH, genomic stability and propensity for aggressive behaviours. For example, tumours can contain cells that have been differentially exposed to acute hypoxia (such as low intracellular O2 levels for minutes to hours) and then reoxygenated. This can reoccur and lead to ‘cycling’ hypoxia. Cells could also be exposed to a more prolonged chronic hypoxia (low intracellu-lar O2 levels for hours to days) before undergoing cell death or reoxygenation. A major concept emerging in clinical oncology is the detection of these tumour cell subpopulations and the determination of their effect both on prognosis and the response to current thera-pies such as surgery, radiotherapy, endocrine therapy or chemotherapy2.

A dynamic tumour microenvironment can not only promote systemic metastasis, but also significantly modify drug action. However, preclinical studies of molecular-targeted agents that inhibit cancer cell signal-ling pathways are rarely conducted under varied micro-environmental conditions (such as within normoxic versus hypoxic conditions) to determine tumour cell kill under conditions that exist in solid tumours. This potential effect of the tumour microenvironment and intratumoural heterogeneity is important in the context of developing individualized cancer medicine and the judicious use of novel molecular-targeted agents.

Transcription and translation in acute and chronic hypoxia. Intratumoural hypoxia is an adverse clinical prognostic factor that is associated with decreased disease-free survival for cancers involving the prostate, cervix, breast, musculoskeletal tissues and the head and neck3–6.

Departments of Medical Biophysics and Radiation Oncology, University of Toronto and Ontario Cancer Institute and Princess Margaret Hospital (University Health Network), 610 University Avenue, Toronto, Ontario, M5G2M9, Canada.Correspondence to R.G.B. e-mail: [email protected]:10.1038/nrc2344Published online 14 February 2008

Hypoxic cellular adaptationCells can continue to proliferate in decreasing O2 gradients by altering the transcription and translation of genes that are involved in angiogenesis and cell invasion, cell metabolism and cell survival.

Hypoxia, DNA repair and genetic instability Robert G. Bristow and Richard P. Hill

Abstract | Areas of hypoxic tumour tissue are known to be resistant to treatment and are associated with a poor clinical prognosis. There are several reasons why this might be, including the capacity of hypoxia to drive genomic instability and alter DNA damage repair pathways. Significantly, current models fail to distinguish between the complexities of the hypoxic microenvironment and the biological effects of acute hypoxia exposures versus longer-term, chronic hypoxia exposures on the transcription and translation of proteins involved in genetic stability and cell survival. Acute and chronic hypoxia might lead to different biology within the tumour and this might have a direct effect on the design of new therapies for the treatment of hypoxic tumours.

h y p o x I a a n d m e ta b o l I s m

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PerfusionTumours have differential perfusion of oxygen and nutrients based on the extent and function of the intratumoural vasculature and interstitial fluid pressure.

Oxygen enhancement ratio(OER). The ratio of radiation dose required for the same biological effect (for example, cell survival or the number of DNA breaks) in the absence versus the presence of O2. This varies for different tumour cell lines and tumour tissues and reflects the radioresistance observed in acutely anoxic cells.

varying durations of chronic hypoxia and/or anoxia can develop in regions of solid tumours owing to reduced intracellular O2 as a function of O2 consumption with increasing distance of the tumour cell from the tumour vasculature. Acute hypoxia and reoxygenation can arise owing to fluctuations in perfusion known to occur within abnormal tumour vasculature (BOX 1, 2).

When tissue metabolism exceeds metabolic supply, as is the case for most tumours, the hypoxic response leads to a series of changes in protein expression to pro-tect against cell death7. Acute cellular hypoxia stabilizes and activates both the ubiquitous HIF1α and the more tissue-specific HIF2α (also known as ePAS1) transcrip-tion factors. This leads to HIF1α- and HIF2α-mediated transcription by bypassing baseline HIF1α and HIF2α degradation, which is mediated by the ubiquitin– proteasome pathway and the von-Hippel lindau pro-tein (vHl)8,9. For example, once stabilized by hypoxia, HIF1α forms a heterodimer with HIF1β (also known as ARNT) on hypoxic responsive elements (HRes) within target gene promoters to drive the expression of HIF1α targets, some well-known examples of which include vascular endothelial growth factor (veGF; involved in angiogenesis), GluT1 (also known as SlC2A; a glu-cose transporter involved in glycolysis) and carbonic anyhydrase IX (CA9; a regulator of cellular pH)7. As such, both HIF1α and veGF are important therapeutic targets to ablate angiogenesis and prevent local and sys-temic resistance to cancer therapy (see REFs 10–14 for recent reviews on these issues). Additionally, novel cell type-specific data from global transcript mining follow-ing hypoxia may give unique prognostic information beyond HIF1α-associated pathways15.

extreme hypoxia or anoxia can also lead to rapid (within hours), but reversible, downregulation of pro-tein synthesis, which is thought to be a means of energy conservation during times of hypoxic stress16–18. There are two distinct pathways leading to this inhibition of translation. The first is rapid, HIF1α-independent, and

mediated by the ‘unfolded protein response’ (uPR). The uPR mediates the phosphorylation of eukaryotic initiation factor 2α (eIF2α) by the endoplasmic reticulum kinase PeRK (also known as eIF2A kinase 3 (eIF2AK3)) leading to inhibition of mRNA transla-tion initiation. The second is a delayed response that is only activated after prolonged (chronic) hypoxia. This response is associated with disruption of the mRNA cap-binding complex, eIF4F, which inhibits the tran-script recruitment step of mRNA translation. A recent study has confirmed that many cell types show severely inhibited mRNA translation in response to anoxia and that disruption of eIF2α phosphorylation can lead to increased cytotoxicity under anoxia19. Indeed, it is remarkable how well living cells adapt to very low O2 conditions. Direct cytotoxicity is usually observed only for tumour cells kept under very low O2 and glucose conditions (such as <0.02% O2) for periods greater than 24 hours, which leads to chromatin condensa-tion, caspase activation and increased apoptosis in a HIF1α-independent manner20.

Current models fail to consistently distinguish between the complex inter-relationships connecting acute hypoxia, cycling hypoxia (acute hypoxia followed by rapid reoxygenation, for example) and chronic hypoxia within the tumour and its effect on differential transcription and translation of proteins involved in genetic stability and cell survival18. even if exposed to similar levels of O2, the duration of a short-term, acute hypoxic exposure versus a longer-term, chronic hypoxic exposure might lead to different biology within the tumour. As we will review below, there is emerging evi-dence that different severities and durations of hypoxia may have different effects on cell cycle checkpoint control and DNA repair and potentially drive genetic mutation and tumour progression4,21–23.

Hypoxic radioresistance and chemoresistance. under normoxic conditions, ionizing radiation (IR) pro-duces DNA double-strand breaks (DNA DSBs), DNA single-strand breaks (DNA SSBs), DNA base damage, and DNA–DNA and DNA–protein crosslinks (DPCs). Classic experiments in vitro and in vivo have demon-strated that, at partial pressure of O2 (pO2) levels below 10 mm Hg, tumour cells can acquire radiobiological hypoxia and hence become relatively resistant to radio-therapy. For example, at 1 mm Hg, they can be 2–3 times more radioresistant than normoxic cells (that is, they have an oxygen enhancement ratio (OeR) of 2–3)24. This occurs because there is decreased fixation of potentially lethal DNA DSBs produced by free radicals following exposure to IR. Therefore, radiosensitization requires the presence of O2 at the time of irradiation. A number of preclinical and clinical assays have confirmed that the number of DNA DSBs that form in anoxic conditions is also decreased by 2–3-fold and that hypoxia can alter the expression and function of DNA DSB-associated genes25–29 (described in more detail below; FIGs 1, 2, 3). By contrast, under anoxia, there is a large relative increase in the number of DPCs that could activate additional DNA repair pathways30.

at a glance

• The presence of intratumoural hypoxia is a negative prognostic indicator for many patients as it has been associated with increased local failure following radiotherapy and increased distant metastatic spread.

• Hypoxia can drive the metastatic phenotype secondary to genetic instability, increased angiogenesis, decreased apoptosis and upregulation of a number of genes involved in the metastatic cascade (such as osteopontin, lysyl oxidase and vascular endothelial growth factor).

• Both acute and chronic hypoxia exist in human tumours and these may have different biological consequences as a function of changes in hypoxia-inducible factor 1α-mediated transcription, altered protein translation and differential activation of hypoxia-associated cell cycle checkpoints.

• Hypoxic cells can acquire a mutator phenotype that consists of decreased DNA repair, an increased mutation rate and increased chromosomal instability.

• Defects in homologous recombination and mismatch repair have been documented in tumour cells that are exposed to chronic hypoxia.

• Defective DNA repair in hypoxic cells could alter the sensitivity to radiotherapy and chemotherapy and render cells susceptible to molecular-targeted agents that are selectively toxic to checkpoint-deficient or repair-deficient tumour cells.

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Interstitial fluid pressure(IFP). High IFP (for example, in the range 10–20 mm Hg) is characteristic many human tumours and is thought to be partly due to abnormal and highly permeable tumour vessels and decreased lymphatic drainage.

2-nitroimidazolesPimonidazole, misonidazole, CCI103F and EF5 are examples of 2-nitroimidazoles that are bioreduced through a series of one-electron reactions in low O2 environments and create stably bound cellular adducts. These O2-dependent adducts can then be detected in vivo using immunohistochemistry or non-invasive imaging.

BleomycinBleomycin is a cytotoxic glycopeptide antibiotic used in chemotherapy. It acts by inhibiting DNA synthesis and creating oxygen-dependent DNA breaks, leading to mitotic catastrophe and cell death.

EtoposideEtoposide is an epipodophyllotoxin that inhibits DNA synthesis by forming a complex with topoisomerase II and DNA. This complex induces DNA double-strand breaks with resulting cell cycle-dependent toxicity (that is, s and G2

phase cells).

Alkylating agentsAlkylating agents (for example, temozolomide, chlorambucil and ifosfamide) add alkyl groups to the bases of DNA, which can lead to DNA breaks and crosslinks and interference with DNA replication and transcription, all resulting in cell death.

CarboplatinCarboplatin is an alkylating agent that covalently binds to DNA to create intra-strand and inter-strand DNA–DNA crosslinks. These crosslinks inhibit DNA synthesis and transcription leading to cell death.

Chronic obstructive pulmonary disease(COPD). A group of diseases, including chronic bronchitis and emphysema, that are characterized by the pathological limitation of airflow in the airway that is not fully reversible.

It is thought that fractionated radiotherapy partially overcomes intratumoural radioresistance because radioresistant hypoxic cells become reoxygenated and therefore resensitized between individual dose fractions. This has led to a large series of preclinical and clinical studies in which tumour radioresistance has been tar-geted with the combined use of fractionated radiotherapy with radiosensitizers or hypoxic cell toxins (for example, 2-nitroimidazoles such as misonidazole or tirapazamine) to increase tumour cell kill and improve overall local control (reviewed in REF. 31). In general, clinical trials that have attempted to decrease tumour hypoxia with these and other drugs have significantly improved local control and overall survival, indicating that for many tumours hypoxia affects treatment outcome despite reoxygenation during fractionated therapy32.

Similarly, hypoxic cells can be chemoresistant for a multitude of reasons, including decreased drug action in the absence of O2 (as is the case for bleomycin and etoposide, for example), the decreased effect of agents in hypoxic cells that are poorly proliferating (cell cycle changes) or have altered pH gradients (such as alkylating agents and anti-metabolites), induction of gene amplification (methotrex-ate) and an overall decreased drug diffusion and delivery to cells distant from functional vasculature33,34. The rela-tive importance of each might depend on the duration of hypoxic exposure (such as acute versus chronic).

More recently, the Dewhirst laboratory has shown that hypoxic gene expression can also modify cellular radioresponse12,35. HIF1α expression during fraction-ated radiotherapy can promote ATP metabolism, proliferation and p53 activation and stimulate endothe-lial cell survival to mediate a final tumour radioresponse. HIF1α-deficient cells may also be more sensitive to carboplatin and etoposide36. This new paradigm has led to the study of novel HIF1α-directed agents as potential chemosensitizers and radiosensitizers12,37.

Resistance and acute versus chronic hypoxia. Within a three-dimensional microenvironment, tumour cells can be exposed to a fluctuating level of O2, which leads to cycling hypoxia for different periods of time. These

fluctuations can lead to differential biology. A caveat to many of the studies that characterize the effect of hypoxia on radiosensitivity and chemosensitivity is that the clonogenic assays in vitro usually mimic the scenario of short-term, acute hypoxia followed by rapid reoxygenation (for example, cells are treated under hypoxia and then placed in an incubator to assay for colony formation 1–2 weeks later while under normoxic conditions). However, cell survival and biology may be different when cells are treated after exposure to longer-duration, chronic hypoxia (that is, greater than 24 h) and maintained under these conditions.

For example, acutely hypoxic tumour cells irradi-ated immediately after reoxygenation are radiosensi-tive when compared with cells irradiated under acute hypoxia alone38. Furthermore, irradiated chronically hypoxic cells (maintained under hypoxia for up to 72 h, for example) can acquire increased radiosensitivity when compared with irradiated cells exposed to acute hypoxia (4–24 h)39,40. The latter studies also showed that radiosensitization with drugs such as misonidazole or SR2508 was increased in chronically hypoxic cells when compared with the toxicity in acutely hypoxic cells (despite the same level of O2 at the time of irradiation). We think that the basis for differential sensitization could be partially explained by the relative ability for DNA repair among cells exposed to short-term versus prolonged hypoxia, as discussed below (FIG. 3).

Hypoxia can drive the metastatic phenotype. The most aggressive manifestation of tumour progression is the development of distant metastases. In many cancer types, patients with hypoxic primary tumours at diag-nosis are more likely to develop recurrence locally as well as at metastatic sites, regardless of whether initial treatment is with surgery or radiotherapy. early clini-cal studies demonstrated that patients with conditions such as anaemia or chronic obstructive pulmonary disease, which were considered likely to induce hypoxia in their tumours, often had a poorer outcome. Although this was thought to be associated with the increased resist-ance of hypoxic cells to radiation (or drug) treatment,

Box 1 | differential hypoxia in human tumours

In tumours, the abnormal vascular architecture and patterning that arises owing to unregulated angiogenesis is an important factor in the development of both chronic and acute hypoxia. Within a three-dimensional microenvironment, tumour cells can be exposed to fluctuating levels of oxygen leading to cycling hypoxia (that is, cycles of acute hypoxia or anoxia followed by reoxygenation) and differential oxygen gradients for different periods of time. These fluctuations can lead to differential biology based on the severity of hypoxia, the duration of hypoxia and whether hypoxia is terminated by cell death or by reoxygenation.

The level of normal tissue oxygenation ranges from 5% (partial pressure of O2 (pO2) of 38 mm Hg), but altered gene expression, biology and radioresistance are consistently reported at hypoxic pO2 levels of <1 mm Hg15,30,103. Chronic hypoxia or chronic anoxia can develop within the three-dimensional environment of tumours as a function of reduced O2 diffusion with increasing distance from the vasculature. Chronic anoxia is due to increasingly long O2-carrying erthyrocyte transit times through the tumour vasculature and an irregular distribution of tumour vessels that leads to limited O2 diffusion within the tumour interstitium at distances >100–200 µm. The duration of exposure to low O2 levels under these circumstances can be prolonged (days) and depends on relative tumour cell proliferation, transit times through the O2 gradient and adaptation to the microenvironment. By contrast, acute hypoxia (exposure to low O2 levels for a duration lasting only minutes to a few hours) followed by reoxygenation in tumours arises because of functional changes in vascular stability and dynamic fluctuations in micro-regional tumour perfusion within tortuous blood vessels. The latter can be further affected by increases in the surrounding interstitial fluid pressure2,22.

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Positron-emission tomography(PET). PET is a non-invasive imaging technique that can create three-dimensional mapping of hypoxia within the body by detecting the γ rays given off by a positron-emitting isotope (for example, 18F).

Single photon emission computed tomography(sPECT). sPECT is a nuclear medicine tomographic imaging technique that the detects γ rays that are given off by radi-opharmaceuticals (for example, 99Tc) and can provide three-dimensional and functional imaging.

Functional computed tomographyFunctional computed tomography (CT) is a non-invasive imaging technique that can measure blood flow, angiogenesis and other physiologic parameters in tissues using image analysis with fast CT scanning and intravenous contrast agents.

Blood oxygenation level-dependent magnetic resonance imaging(BOLD MRI). BOLD MRI is a non-invasive technique for indirectly measuring tissue perfusion and oxygenation based on the paramagnetic qualities of deoxyhaemoglobin. BOLD MRI does not require exogenous contrast materials and one can obtain serial images at high spatial resolution over time.

Polarographic O2 electrode measurementsA microelectrode needle can be placed into normal or tumour tissue and can determine partial pressure of O2 (pO2) measurements as it moves forward through the tissue. This can be used to compare tumour hypoxia within a cohort of patients.

there was also evidence that this remained a factor even for patients undergoing surgical treatment. These issues came into clearer focus with the publications of Hockel et al. and Brizel et al. in 1996, which demonstrated, using polarographic O2 electrode measurements, that patients with more hypoxic tumours did indeed have poorer outcomes, at least partly owing to an increased propensity to develop metastatic disease3,41.

These clinical studies have been supported by extensive experimental work initiated by the observa-tions by Young et al.42,43 that ex vivo or in vivo expo-sure of rodent tumour cells to hypoxia can increase experimental or spontaneous metastases (BOX 3). These early experiments supported chromosome over-replication and gene amplification as one fac-tor in driving the acquired metastatic phenotype. In both tissue culture and animal models, hypoxia has also been associated with gene amplification, point mutation, hyper-mutagenesis and induction of DNA strand breaks during acute hypoxia and reoxygena-tion44–46 (see also section on genetic instability below). If not repaired, these breaks and mutations could activate oncogenes or inactivate tumour suppressor genes resulting in the selection of tumour cell variants with increased growth and metastatic potential.

Additional studies with human melanoma and other tumour cells suggested that hypoxia could drive the metastatic phenotype by upregulating genes involved in the metastatic cascade - such as plasminogen activator urokinase receptor (PLAUR), the chemokine receptor CXCR4, osteopontin (also known as secreted phos-phoprotein 1 (SPP1), lysyl oxidase (LOX), interleukin 8 (IL8) and VEGF — mostly as a result of increased HIF1α expression47-49. These changes in gene expression are usually transient with fairly rapid decay back to normal

levels when the exposure to hypoxia ceases. Recent studies have demonstrated a role for the expression of LOX both in the initiation and growth of metastases in a rodent model system, possibly associated with maintenance of the postulated metastatic niche derived from bone marrow-derived haematopoietic progenitor cells50. High expression of LOX has also been linked to increased metastasis in patients49.

It has also been reported that exposure to hypoxia can select for cells with loss of p53 function and increased ability to withstand apoptotic stimuli51. The concept that hypoxic exposure can increase resistance to apoptosis is consistent with our own findings that, in some rodent cell lines, hypoxia increases expression of the p53-negative regulator MDM2 and this leads to increased resistance to apoptosis in vivo and increased metastasis formation52. More recently we have demonstrated that human HT-1080 cells show similar resistance to apoptosis in vivo following hypoxic exposure53. These findings, however, are caused by transient changes in gene expression, and not selection of cells with a pre-existing resistance to apoptosis, as in the original studies of Graeber et al.54. Additionally, whether p53 status controls hypoxia-induced metastasis remains to be proven clinically — at least one study of soft-tissue sarcomas did not find an association between hypoxia, p53 mutation and clinical outcome55.

An important question is whether exposure to acute or chronic hypoxia has a more important role in the induction of the increased metastatic potential of cells and whether this is also dependent on the severity of hypoxia. Although (as described in BOXEs 1, 2) it should be noted that in situ the temporal boundaries of these multiple states in tumours are ill-defined, our work demonstrated that deliberate exposure to defined cycles of hypoxia in vivo could increase the

Box 2 | measuring hypoxia in tumours

The intrinsic hypoxic cell biomarkers of the hypoxia response can include immunohistochemical (IHC) detection of hypoxia-inducible factor 1α (HIF1α), vascular endothelial growth factor (VEGF), carbonic anyhydrase IX (CA9) or GLUT1 proteins and measurement of levels of plasma osteopontin (a non-collageneous matrix protein). Although the IHC markers give information about hypoxia with regard to the tissue architecture, they do not measure absolute O2 concentrations as HIF1α targets are subject to the complex regulation of HIF1α itself, which is also regulated by many factors in addition to O2. However, CA9 and GLUT1 have been proposed as markers of prolonged chronic hypoxia because long exposure to low levels of O2 can increase their expression. At present, no individual intrinsic marker can differentiate between acute and chronic hypoxia. Extrinsic hypoxic cell biomarkers include systemically administered 2-nitroimidazoles (such as pimonidazole, EF5 and CCI-103F) which, at pO2 levels below 10 mm Hg, undergo hypoxic bioreduction to create irreversible intracellular adducts that are detectable by specific antibodies. These biomarkers can give a quantitative measurement of the level of O2. Non-invasive imaging techniques for hypoxia include the use of radiolabelled 2-nitroimidazoles and other probes imaged with positron-emission tomography ([18F]misonidazole, [18F]fluoroazomycin, 18F-EF5 and 60Cu-ATSM), single photon-emission computed tomography ([123I]iodoazomycin), functional computed tomography and magnetic resonance imaging, and BOLD (blood oxygenation level-dependent) magnetic resonance imaging. Functional imaging provides anatomical information in addition to tumour perfusion and vascular permeability2,103.

An experimental protocol has been developed in which sequential administration of two extrinsic markers within 2–3 h of each other (pimonidazole followed by CCI-103F, for example) allows for the relative determination of chronic hypoxia to acute hypoxia. In this case, co-localization of the second marker to the first will only occur in chronically hypoxic regions. By contrast, acute hypoxia is reflected by mismatches in the two biomarkers in which vessel perfusion was acutely altered during the period between serial injections103. Given the potential differences in biological behaviour between acute and chronic hypoxia, it will be important to develop similar, but clinically applicable, techniques using non-invasive imaging or panels of biomarkers to determine the relative fraction of acute to chronic hypoxia and absolute O2 concentrations as potential prognostic and predictive biomarkers.

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(to quantitate DNA DSB numberand protein–proteinco-localization)

Nature Reviews | Cancer

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development of metastasis in both a rodent and human xenograft model system44,56 (BOX 3). Similarly, recent studies by Rofstad et al.23 using human melanoma xenografts support the concept that acute hypoxic exposure might have a more important role than chronic hypoxia in metastasis induction. Reasons for this are currently unclear5, but might depend on dif-ferential levels of O2 between different model systems. Comparison studies of the effects on gene expression of acute versus chronic exposure, in addition to docu-menting potential stable genomic changes, are urgently needed to address this issue. exposure to cycling hypoxia can lead to increased levels of reactive oxygen species (ROS) in the cell, in part owing to increased release of such species from mitochondria57–59. ROS

are capable of causing DNA damage and could drive malignant progression, a hypothesis that is currently being investigated (see below).

Genetic instability in hypoxic cellsGiven that many human tumours contain hypoxic sub-populations, it is surprising that there are few data con-cerning the sensing and repair of DNA damage under experimental conditions of acute cycling, or chronic hypoxia. experimental evidence to date suggests that hypoxia and anoxia can indeed alter cell cycle checkpoint control and/or the sensing and repair of DNA damage.

Hypoxia can activate ATM and ATR checkpoints. DNA damage-associated checkpoints normally protect against carcinogenesis as a means to repair potentially mutagenic or sublethal DNA damage. Anoxia can induce a G1 and intra-S phase (DNA replication) arrest. A further G2 phase arrest can ensue upon reoxygenation. These checkpoints are controlled by the WAF1–Cyclin e–CDK2 (cyclin-dependent kinase 2), ATM–p53–CHK2 and ATR–CHK1 pathways, and the phosphorylation of BRCA1 (REFs 60–62). A recent paper also suggests that anoxia can suppress the expression of CDC25A (cell division cycle 25A), also preventing S-phase progression63.

Many factors could alter overall DNA repair and cell fate following endogenous or exogenous DNA damage in a solid tumour under hypoxic condi-tions, including the relative percentage of normoxic or hypoxic cells (and whether the latter are acutely or chronically hypoxic), the resultant gene expression and protein function at a range of low O2 levels that exist in human tumours (such as differential gene and protein expression between 0.0–5.0% O2) and the duration or cell transit times of cells through normoxic–hypoxic–anoxic gradients. Hypoxia or anoxia may or may not always cause DNA damage during S-phase, but it has been suggested that reoxygenation produces ROS that generate DNA damage (DNA SSBs and possibly DNA DSBs) and this elicits a CHK2-dependent G2 check-point. Indeed, CHK2-deficient cells do not undergo G2 arrest following anoxia and instead undergo apopto-sis60,61. By contrast, chronic hypoxia leading to cellular adaptation may not activate G1 or S checkpoints and could potentially lead to accumulation of DNA replica-tion errors or DNA breaks over time18 (FIG. 4). However, whether it is primarily the level of residual O2, the dura-tion of hypoxia or anoxia, or the reoxygenation events per se that determines whether cell cycle progression is affected within the complexity of a solid tumour has not been adequately addressed in vivo. It remains to be determined whether cycling hypoxia or chronic hypoxia can be directly linked to tumour progression (that is, hypoxia-induced metastases) through altered ATM and/or ATR checkpoints.

Hypoxia might lead to increased rates of mutation. The altered checkpoint control indicates that hypoxic cells could acquire a mutator phenotype. Several laboratories have reported increased mutagenesis using mutation reporter constructs in cells exposed to in vitro or in vivo

Figure 1 | Dna double-strand break sensing and repair pathways. Under normoxic conditions, a DNA double-strand break (DNA DSB) is sensed by the MRE11–RAD50–NBS1 (MRN) complex (a). This leads to activation and recruitment of the ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKCS) kinases and phosphorylation of the histone variant H2AX (termed γH2AX, yellow) around the site of the break. Subsequently, a number of DNA damage sensing proteins (such as mediator of DNA damage checkpoint 1 (MDC1) and p53-binding protein 1 (53BP1)) and DNA DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ) are recruited within the first 1–6 h of damage recognition to repair the DNA DSB. The NHEJ repair pathway can be used within any phase of the cell cycle and can be error-prone. The HR pathway is preferentially active in the S and G2 phases of the cell cycle when a homologous sister chromosome or chromatid is available for direct base-pairing to effect error-free repair of a DNA DSB66,102. The induction, resolution and co-localization of DNA damage sensing and repair proteins can now be quantified using immunofluorescent microscopy and image analysis to ‘count’ nuclear DNA repair foci at the sites of DNA damage at a given time following genotoxic insult (b). Shown on the left is a normal diploid fibroblast stained for γH2AX foci (green) in two dimensions and three dimensions at 30 min after a dose of 2 Gy. Both approaches can be used to quantify DNA DSBs and three-dimensional approaches are particularly useful for co-localization approaches (here showing γH2AX and 53BP1 (red)) within the nuclear volume. BLM, Bloom syndrome; EME1, essential meiotic endonuclease 1 homolog 1 (S. pombe); RPA, replication protein A.

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ca Normoxia (21% O2)b Normoxic nucleus (G1 phase)

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hypoxic conditions; these data are consistent with the hypothesis that the hypoxic environment can be hyper-mutagenic. Reynolds et al.64 used a chromosomally inte-grated reporter gene within a λ shuttle vector to show a 5-fold increase in mutation frequency for cells grown in tumours compared with cells grown in culture. using an enhanced green fluorescent protein reporter gene and a genomic mini-satellite locus, li et al.65 also showed an

increased mutation frequency following growth of 4T1 mouse cells in vivo as subcutaneous tumour implants in syngeneic mice. using a mutation rodent model in vivo, Papp-Szabo et al.45 showed a 2-fold increase in mutation frequency as measured by a transgene construct in mam-mary epithelial cells following 24 h hypoxic exposure or when transformed mammary cells were grown as xenografts.

Figure 2 | Dna double-strand breaks and chromatin in irradiated hypoxic cells. a | The number of initial γH2AX foci, which are thought to represent DNA double-strand breaks (DSBs), is dependent on the O2 concentration at the time of irradiation. In general, anoxic cells will have 2–3 times fewer DNA DSBs than normoxic cells for a given radiation dose. Shown is the O2 dependency for γH2AX formation following exogenous DNA damage in G0–G1-synchronized fibroblasts at 30 min after a dose of 2 Gy. From top to bottom, cells were irradiated under 21%, 0.2% or 0.0% O2 gassing conditions. Note also the change in chromatin structure based on DNA 4′,6-diamidino-2-phenylindole (DAPI; blue) staining in the anoxic cells. The biology underlying these changes is unclear but might represent chromatin remodelling and altered heterochromatin or euchromatin responses under low O2 conditions and is an active subject of study in the authors’ laboratories (R. Kumareswaran & R.G.B., unpublished observations). b | Under non-irradiated normoxic conditions, the γH2AX and p53-binding protein 1 (53BP1) DNA DSB responses will not be activated into discrete foci within the nucleus in proliferating cells. However, under non-irradiated anoxic conditions, the ataxia telangiectasia mutated (ATM) kinase can be activated and associated with an intra-S-phase checkpoint and γH2AX can be induced throughout the chromatin. Shown here is a normal diploid fibroblast nucleus with pan-53BP1 staining (red) and minimal γH2AX foci (green) under non-irradiated normoxic conditions in G1 phase (upper panel) and a non-irradiated anoxic fibroblast nucleus arrested in S-phase (lower panel) with global γH2AX activation (green) throughout the chromatin. (To view three-dimensional video representations of these nuclei see Supplementary information S1, S2 (movies)). c | One can also potentially use DNA repair foci to track DNA DSBs and their repair within solid tumours. In the example shown, 22RV1 prostate cancer xenografts were stained for the extrinsic hypoxic biomarker EF5 (red) and the DNA DSB response biomarker 53BP1 (green) at 24 h following irradiation in vivo with five daily fractions of 2 Gy. Sections were visualized using spinning disk confocal microscopy. The normoxic to hypoxic gradient can be visualized as a decrease in EF5 staining from lower left to upper right of the tumour histology section. In this case, there is an increase in residual DNA DSBs within the normoxic portion of the section (indicated by increased 53BP1 nuclear foci) when compared with the EF5-avid portion of the section. Whether the EF5-positive section contains solely acutely hypoxic cells or chronically hypoxic cells is not known and would require co-staining with selective biomarkers for each type of hypoxia as described in BOX 2.

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Studies by Sandhu et al.66 demonstrated that the tumour environment and the presence of neutrophils caused mutations that could be abrogated by treatment of the mice with vitamin e. Similarly, we observed that there were increased DNA breaks in cells from tumours in mice exposed to cycles of low O2 breathing, which could be reduced by treating the mice with euK-189, an agent with superoxide dismutase–catalase (CAT) activ-ity (R. Cairns and R.P.H., unpublished observations). Hammond et al.62 also used a comet assay to demon-strate that reoxygenation induced significant amounts of DNA damage and that this was due to the presence of ROS during reoxygenation. Phosphorylation of serine 15 of p53 and phosphorylation of histone H2AX (also known as γH2AX) was initiated by ATR under hypoxia but was maintained by ATM during reoxygenation62

By contrast, more recently Banath et al.67 have shown that mutation frequency and DNA SSB and DNA DSB repair might not be significantly affected in hypoxic subpopulations of a veGF-overexpressing v79

Chinese hamster xenograft. Despite showing increased radioresistance, ex vivo hypoxic cells sorted by flow cytometry from solid tumours did not have increased mutations in hypoxanthine phosphoribosyltrans-ferase 1 (HPRT1) or defective DNA DSB repair kinetics (as measured by the comet assay and γH2AX staining). However, these experiments only addressed the repair of DNA DSBs in cells irradiated under hypoxic conditions followed by DNA DSB repair under reoxygenated condi-tions. Whether the same observations would be true for cells irradiated and kept under continual hypoxia dur-ing repair, mimicking conditions of prolonged hypoxic exposure in vivo, is not yet known.

Hypoxia and DNA repair. Human DNA DSBs are usu-ally repaired through two main pathways: homologous recombination (HR) and non-homologous end join-ing (NHeJ)68–70. These pathways vary in their protein components, their cell cycle specificity and their fidel-ity of repair (FIGs 1, 2). In studies of normoxic cells,

Figure 3 | Hypoxia decreases raD51 and homologous recombination. a | Exogenous and endogenous (replication-associated) DNA double-strand breaks in the S and G2 phases of the cell cycle are repaired mainly through homologous recombination (HR)70 (FIG. 1). Tumour cells defective for HR exhibit increased chromosomal instability. Cells gassed under 24–72 h of chronic hypoxia that allows for continued proliferation have lower RAD51 protein levels in the HR-sensitive S and G2 phases of the cell cycle than proliferating normoxic cells. This leads to compromised HR function when compared with proliferating normoxic cells. Also shown is data supporting the assertion that there is a smaller effect on RAD51 levels following treatment with cobalt chloride, CoCl2, which induces hypoxia-inducible factor 1α (HIF1α). These and other data using isogenic cell lines for HIF1α expression82 have confirmed that downregulation of HR expression under hypoxia and anoxia is HIF1α-independent. Decreased HR function in chronically hypoxic tumour cells could lead to increased sensitization using agents that are preferentially toxic to HR-defective cells (such as mitomycin C, cisplatin, etoposide or inhibitors of poly (ADP-ribose) polymerase 1 (PARP1)). Part a modified, with permission, from REF. 83 (2005) Elsevier Science. b | The graph shows an example of how chronically hypoxic tumour cells can have differential sensitivity to DNA damage (ionizing radiation (IR)) when compared with acutely hypoxic cells. Chronically hypoxic human lung cancer H1299 cells rendered defective in HR by 72 h of prolonged gassing at 0.2% O2 are not as radioresistant as the same cells gassed for 6 h (acute hypoxia), despite irradiation under the same O2 concentration. Indeed, pretreatment with hypoxia for 72 h followed by normoxic radiation (mimicking prolonged hypoxia followed by reoxygenation) sensitizes the cells to ionizing radiation to an even greater extent. This finding is similar to the relative IR-sensitivity of HR-defective normoxic cells (such as cells defective in BRCA1 and BRCA2 relative to HR-proficient controls). Owing to the fact that the turnover of chronically hypoxic cells may be shorter than 72 h in vivo (depending on tumour type and competing cell death), these in vitro data will require confirmation with complementary studies in solid tumours in vivo. Part b modified, with permission, from REF 77 (2008) American Association for Cancer Research.

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Day 14: Lungs examined for spontaneous metastases

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Fanconi anaemiaFanconi anemia is an autosomal recessive disorder that leads to aplastic anaemia and increased sensitivity to DNA-damaging agents. The disorder is caused by altered activity of one of at least 13 genes that encode the FANC proteins that function within a pathway as part of the DNA damage response.

Nucelotide-excision repairThis pathway recognizes bulky distortions in the DNA that occur after ultraviolet radiation or chemotherapy. Recognition of these distortions leads to the removal of a short single-stranded DNA segment that includes the lesion, creating a single-strand gap in the DNA, which is subsequently filled by a DNA polymerase.

the inhibition of the HR and NHeJ repair pathways has been linked to increased genetic instability and carcinogenesis71–76. Therefore, maintaining error-free HR under normoxic and hypoxic conditions could be important to preserve genomic stability to prevent a mutator phenotype, but might also alter sensitivity to DNA-damaging agents77,78

The concepts surrounding hypoxia, altered spec-tra of DNA damage and defective DNA repair start to converge when one studies the relative hypoxic radioresistance in cells that are defective in a variety of DNA repair pathways. Cells with defects in the HR repair pathway and cells derived from patients with Fanconi anaemia have increased sensitivity to IR as well as decreased OeRs (for example, OeRs of 1.5–2.0 compared with OeRs of 2.6–3.0 for repair-proficient cells)79. In the same study, no change in OeR was noted for NHeJ-deficient cells. In other work, the ERCC1 and nucleotide-excision repair (NeR) pathway have been implicated in hypoxia-mediated radioresistance, pos-sibly through ineffective repair of IR-induced DPCs under hypoxic conditions80,81.

Despite the importance of DNA repair in genomic stability, few papers studying the effect of hypoxia on the specific HR or NHeJ repair pathways in detail have been published. Recent studies from our laboratory and that of Peter Glazer have supported the concept that HR may be compromised in cells exposed to hypoxic conditions. Our joint initial studies suggested that RAD51 expression was reduced by periods of hypoxia

greater than 24 h both in vitro and in vivo. This obser-vation was made across multiple cell histopathologies in a HIF1α- and cell cycle-independent manner82. The latter is important as it was consistent with an effect of hypoxia on the expression of proteins involved in HR that was not linked to altered fractions of cells in the S and G2 phases of the cell cycle, where RAD51 expres-sion is maximal. Furthermore, RAD51 levels remained decreased for periods of up to 48 h after reoxygena-tion82. In further studies with a series of prostate cell lines, we confirmed that the hypoxia-mediated decrease in the expression of RAD51, RAD54, BRCA1, BRCA2 and the RAD51 paralogues was not associated with p53 status, relative capacity for hypoxia-induced apoptosis or cell cycle distribution of the cell lines tested83.

Bindra and colleagues have also studied the effects of hypoxia on NHeJ and HR using a plasmid reporter system. They observed that the frequency of lesions repaired by HR was decreased with hypoxia; there were no decreases in NHeJ activity77,84. This result was also confirmed in our laboratory using restriction-enzyme-induced breaks and a flow-cytometric repair assay that measures high-fidelity HR. These multiple studies also confirmed that the decrease in HR activity is HIF1α-independent by studying the expression of HR proteins in cells isogenic for HIF1α function and cells treated with the HIF1α-inducing agent, cobalt chloride82,83. Together these data suggested that the effect on HR proteins is observed under conditions of low O2 lev-els and might not always be replicated with the use of

Box 3 | acute hypoxia can increase reactive oxygen species and metastasis in vivo

Experimental and clinical evidence supports the assertion that intratumoural hypoxia can increase both experimental and spontaneous metastases. The relative effect of chronic hypoxia versus acute hypoxia and reoxygenation on this process can be tested using cycling hypoxia experiments41,48,49. Tumour-bearing animals are randomly assigned to either control, chronic hypoxia (2 h breathing 7% O2) or cycling hypoxia (12 cycles of 10 min 7% O2 followed by 10 min air) daily and then evidence for metastasis is scored. In the data shown for the KHT sarcoma model, the cyclic hypoxia regimen increased spontaneous lung metastases more than the chronic hypoxic regimen did. Figure reproduced, with permission, from REF. 56 (2001) American Association for Cancer Research.

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Clonal selection• Aggressive tumour cell phenotypes

Increased metastasis• Poor clinical prognosis

Acute hypoxia(minutes to hours)• HIF1α activation

Increased genomic instability• Residual DNA damage• Defective repair• Chromosomal aberrations/fragility and aneuploidy

Increased resistance to radiotherapy and chemotherapy

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Decreased DNA repair (HR, MMR, BER)

Reoxygenation• Increased ROS • Reversible G2 checkpoint

Potential bypass of ATM and ATR checkpoints Proliferation

Increased sensitivity to radiotherapy, chemotherapy and HR-targeted agents (such as anti-PARP)

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ration

HIF1α-mediated expression of metastatic genes (VEGF, LOX)

chemicals that solely mimic hypoxic transcriptional responses (FIG. 3).

The data concerning the effects of chronic hypoxia on NHeJ are mixed. Gene expression studies conducted by Meng et al.83 showed downregulation of many mRNA species encoding proteins associated with NHeJ following chronic hypoxia (72 h of 0.2% O2), but there was no change in protein expression. A contrasting report by um et al.85 suggested that acute hypoxia (4 h of 1% O2) could upregulate the expression of Ku70 (also known as XRCC6)–Ku80 (also known as XRCC5) and DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKCS, also known as PRKDC) as well as DNA-PK activity in mouse and human hepatoma cells (HepaC1C7 and HepG2 cells, respectively). This same group demonstrated that DNA-PK could directly interact with HIF1α and that DNA-PKCS might be involved in phosphorylation of HIF1α. This implies that the increased expression or activation of DNA-PK under hypoxic conditions might contribute to the stabilization of HIF1α; however, additional experi-ments are required to assess whether this leads to a functional increase in NHeJ. Hypoxia has also been shown to decrease the expression of NBS1 (also known

as nibrin), which is part of the MRe11–RAD50–NBS1 (MRN) complex that initially recognizes DNA DSBs, leading to the induction of γH2AX foci (which may be indicative of DNA DSBs). This downregulation of NBS1 occurs in a HIF1α-dependent manner and requires thre-onine phosphorylation of the PASB (Per–ARNT–Sim B) domain in the HIF1α protein86.

In addition to DNA DSB repair and NeR, another important pathway that can be modified by hypoxia is the mismatch repair (MMR) pathway. MMR normally corrects DNA base pair mismatches during DNA rep-lication and when defective gives rise to microsatellite instability. Hypoxia appears to induce the downregula-tion of two MMR genes, MLH1 and MSH2, leading to increased mutagenesis and dinucleotide repeat insta-bility; this was also associated with hypoxia-mediated alterations in histone deacetylation21,87–89. This finding might have implications for the sensitivity of hypoxic cells to alkylating agents (such as temozolomide, which shows clinical activity for the treatment of patients with glioma) suggesting that hypoxia-mediated decreases in MMR could also increase resistance to some agents.

Note that HIF1α and HIF2α might have differential biology in DNA repair90,91. At the biochemical level,

Figure 4 | Model of hypoxia-mediated genetic instability. Cells exposed to cycling hypoxia (acute anoxia followed by reoxygenation) may activate ataxia telangiectasia mutated (ATM)–ATR (ataxia telangiectasia- and Rad3-related kinase)-mediated cell cycle checkpoints to arrest the cell in order to repair any DNA damage caused by reactive oxygen species (ROS). Non-repaired DNA breaks in proliferating cells could give rise to genetic instability and clonal selection of aggressive tumour mutants, resulting in metastasis. Acute hypoxia can also increase the expression of hypoxia-inducible factor 1α (HIF1α) and upregulate genes involved in angiogenesis and metastasis (such as vascular endothelial growth factor (VEGF) and lysyl oxidase (LOX)). Cells exposed to prolonged chronic hypoxia can also acquire genetic instability through decreased translation of DNA repair proteins leading to defective repair in proliferating cells and an increased mutation rate. As shown, hypoxia could increase or decrease the sensitivity of tumour cells to radiotherapy or chemotherapy depending on the level and duration of hypoxia and the molecular pathways that are affected by the hypoxic response. BER, base-excision repair; CHK2, checkpoint kinase 2; HR, homologous recombination; MMR, mismatch repair; PARP, poly (ADP-ribose) polymerase.

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Polysomal fractionation analysisThis technique can determine the mRNA translation efficiency of specific genes following cellular hypoxia or other stressors.

the work by Koshiji et al.88 showed that intracellular hypoxia drives HIF1α to displace MYC from the MSH2 gene promoter, thereby decreasing MSH2 transcription and increasing microsatellite instability. This important finding by the Huang laboratory was instrumental in defining a potential role for MYC in maintaining genomic stability. Further work from this laboratory has reported HIF1α-mediated changes in proteins involved in DNA damage sensing and checkpoints, including WAF1, NBS1 and CDC25A (reviewed in REF. 91). In complementary studies, Bindra and Glazer89 proposed another model of MYC biology under hypoxic condi-tions, in which hypoxic cells have decreased MYC expression and decreased binding to the promoters of MLH1 and MSH2. The decreased binding was sec-ondary to a relative shift from activating MYC–MAX (MYC-associated factor X) heterodimers to repres-sive mitotic arrest deficient-like 1 (MAD1l1)–MNT complexes at the MLH1 and MSH2 promoters. Finally, a recent report suggests that, in contrast to HIF1α antagonizing the activity of MYC, HIF2α can enhance MYC activity and augment MYC transformation90. Altogether, these studies support an interplay between positive and negative influences in MYC-mediated cellular transformation under normoxic and hypoxic conditions91.

Transcription or translation as a factor in altered DNA repair? In addition to the effect of hypoxia activating and repressing transcription factor complexes in rela-tion to MLH1 and MSH2 gene expression, Bindra and Glazer have also documented hypoxia-induced transcriptional repression on Rad51 and Brca1 gene expression21,78. In this case, hypoxic downregulation of the Rad51 and Brca1 genes is mediated by altered e2F transcriptional activation and repression. In other cell lines, the RNA and protein expression of HR genes was discordant after chronic hypoxic exposure77,83, sug-gesting an additional level of genetic control. Indeed, a complementary model of hypoxia-induced translational repression has been proposed19,92 and could explain decreased HR expression under hypoxia. In recent work, using polysomal fractionation analysis of RAD51 and BRCA2 transcripts in collaboration with the labo-ratory of Brad Wouters and Marianne Koritzinsky, we have observed that the expression of these genes can be downregulated at the translational level by hypoxia77 and leads to reduced HR (FIG. 3). It seems that both altered transcription and translation can be important in determining hypoxia-mediated changes in DNA repair protein expression. The relative contribution of one over the other might vary as a function of cell type or cell line, O2 concentration and experimental design, and requires isogenic cell systems to explore the role of differential translational control.

Modification of chromatin in hypoxic cells. A power-ful argument supporting the hypothesis that hypoxia can drive genetic instability stems from experiments that documented induction of common fragile sites in hypoxic cells. These sites are highly unstable in the

human genome and are prone to chromosomal break-age and rearrangement93,94. In one study, the hypoxia-induced fragile site was shown to drive the formation of double minute chromosomes and their widespread insertion as homogenously staining chromosomes, all cardinal features of gene amplification. These inde-pendent experiments connect satisfactorily to the early observations of Young and Hill, who linked hypoxia-mediated metastasis to DNA over-replication and gene amplification42,95. Interestingly the studies of Young et al.42 suggested that over-replicated cells could convert back to their usual hyperdiploid state during further growth under reoxygenated conditions. Hypoxia can also increase telomerase activity in cells in a HIF1α-dependent manner96,97, although the implications for cellular senescence and transformation through altered telomere biology has not yet been explored. Other chro-matin responses to hypoxia include global deacetylation and methylation of histones, phosphorylation of γH2AX and premature condensation of chromosomes and abnormal chromosome mis-segregation98,99.

Whether continual long-term culture of immortal-ized cells under normoxic versus hypoxic conditions can transform cells with unique clonal karyotypes that reflect altered DNA repair awaits further study (FIG. 4). Such experiments are required to directly connect defective DNA repair and increased mutagenesis under hypoxic conditions with carcinogenesis and, ultimately, tumour progression.

targeting genomic instability in hypoxic cellsWith the knowledge that hypoxic cells confer a negative prognosis, it is imperative that these cells are targeted during cancer therapy. It would seem intuitive that a strategy for chemoprevention would include the use of antioxidants to prevent production of ROS and DNA damage created during cycling hypoxia. This could pre-vent genetic instability and the transformation process. The concept of reducing ROS-associated genetic insta-bility has been an important concept in chemopreven-tion strategies relating to prostate and other cancers as it might inhibit cellular carcinogenesis100.

However, a recent study101 has shown that antioxi-dants may prevent cancer progression through inhibi-tion of HIF1α-mediated angiogenesis rather than solely inhibition of genetic instability. In three tumorigenic models in vivo, the anti-tumour efficacy of antioxidants was linked to diminished HIF1α activity. ectopic expression of an O2-independent, stabilized HIF1α mutant rescued lymphoma xenografts from inhibition by two antioxidants: N-acetylcysteine and vitamin C101. As HIF1α also regulates the transcription of many genes involved in immortalization, maintenance of stem cell pools, cellular de-differentiation, genetic instability, vascularization, metabolic reprogramming, invasion and metastasis, and radiotherapy treatment failure, these data add additional support to the development of novel therapies that target HIF1α10–12.

The induction of DNA damage in tumour cells by chemotherapy and radiotherapy is a mainstay of cur-rent cancer treatment. Fractionated radiotherapy has

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Base-excision repairBase-excision repair uses DNA glycosylases and AP endonucleases to remove DNA bases (caused by exogenous or endogenous ROs and alkylation) in order to prevent cytotoxicity or DNA polymerase errors. Once the damaged DNA base is removed, DNA polymerase and ligase activities regenerate the DNA strand and seal the DNA.

been successful in many tumour types as it is thought that fractionation leads to tumour reoxygenation and optimal repair in normal tissue to derive a favourable therapeutic ratio (that is, increased killing of tumour cells relative to normal tissues)102. Although often thought to be offset by reoxygenation, intratumoural hypoxia is still viewed as an important cause of radio-therapy failure, which requires implementation of novel clinical strategies32. This includes the development of drugs — such as tirapazamine and PR-104 — that can directly target poorly oxygenated cells32,103. Indeed, the mechanism of cell kill of these hypoxic cell toxins can involve the production of DNA breaks and DPCs and tirapazamine requires the HR pathway for its hypoxic cytotoxic effects104.

This, taken together with the data that supports reduced DNA repair capacity for cells exposed to chronic hypoxia, implies targeting hypoxic tumour cells that have defective DNA repair capacity might be a further strategy to improve the therapeutic ratio. Indeed, our recent work confirms that chronically hypoxic cells might be more sensitive to IR, mito-mycin C and cisplatin than acutely hypoxic cells77. Differential repair under different hypoxic condi-tions might also be one factor in the relative success of radiotherapy in effecting local control, as chroni-cally hypoxic cells might not be as radioresistant as suggested by earlier in vitro studies, which used acute hypoxic exposures to analyse the effects of hypoxia on radiation response. However, these in vitro findings must now be confirmed in vivo because chronically hypoxic cells may exist to different extents in tumours of varying histopathology owing to different durations and degrees of O2 restriction.

Furthermore, malignant hypoxic tissues may have discordance between cell cycle checkpoint control and DNA repair. This could be exploited using agents such as uCN-01, CeP-3891, roscovitine and flavopiridol, which target the CHK2–CHK1 pathways, or PRIMA1 or nutlin compounds that target mutant p53 and abnor-mal MDM2 regulation105. Indeed, we have observed that both PRIMA1 and nutlin 3 can sensitize tumour cells under normoxic and hypoxic conditions (S. Supiot, R.G.B. and R.P.H., unpublished observations).

In thinking about additional ways to target chroni-cally hypoxic cells, one can use the concept of synthetic lethality, whereby inhibition of a final survival path-way in a DNA repair-defective tumour cell leads to cell toxicity (FIG. 4). For example, tumour cells derived from patients with BRCA1 and BRCA2 mutations lack functional BRCA1 and BRCA2 proteins and are defec-tive in HR. Poly(ADP-ribose) polymerase 1 (PARP1) facilitates base-excision repair by binding to DNA SSBs. PARP1 inhibitors such as 3-AB, ISQ, Nu1025, Ku0058684 or AG14361 can act as radiosensitizers in vitro and in vivo and trigger γ-H2AX and RAD51 foci formation106.

Recently, it has been observed that BRCA1 or BRCA2 deficiency profoundly sensitizes cells to the inhibition of PARP1 enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis as

a result of persisting DNA lesions that would normally be repaired through HR107,108. This novel result could be clinically exploited to selectively kill BRCA2-deficient tumours by using PARP1 inhibition, in combination with chemotherapy or radiotherapy. Furthermore, Ashworth and coworkers109, using a small interfering RNA approach, observed sensitivity to PARP1 inhi-bition in a variety of cells made deficient in proteins involved in the HR or Fanconi anaemia pathway (such as RAD51, RPA (replication protein A), NBS1, ATR, ATM, CHK1, CHK2 and FANCD2 (Fanconi anaemia complementation group D2)). Similarly, a recent report suggests that cells deficient in the Fanconi anaemia pro-teins are more sensitive to inhibition of ATM; indeed small-molecule inhibitors of this kinase are being stud-ied in preclinical settings and might soon be available clinically102,105,110. Many of these proteins are deregulated in cancer and/or downregulated by hypoxia83. Indeed, preliminary evidence from our laboratory suggest that chronically hypoxic cells, but not acutely hypoxic cells, are sensitive to PARP1 inhibition (N. Chan, S. liu and R.G.B., unpublished observations).

Concluding remarksThere are a number of outstanding questions to be answered in making direct links between hypoxia, genetic instability and aggressive phenotypes. These include the need for direct evidence in a premalignant model that hypoxia leads to defective DNA repair that permanently alters genomic stability and leads to cel-lular transformation (FIG. 4). Similar data is required to establish that in existing cancer cells hypoxia perma-nently, rather than transiently, alters protein expression through genomic changes to drive increased cell prolif-eration, cell survival and the metastatic phenotype.

Another major area of research that is needed to enable effective translation of these concepts into the clinic is in new techniques that can quantify and moni-tor acute versus chronic hypoxia to provide selection of individualized cancer therapies103. Given the possibility of hypoxia-mediated alterations in transcription and translation, it is also desirable to test the efficacy of new molecular-targeted agents under normoxic and hypoxic (both acute and chronic) conditions. establishing that the targeted molecules or pathways are inhibited equally under both scenarios will help to ensure that clinical resistance is not solely due to a drug that is ineffective in hypoxic cells, although limited drug diffusion could still confer survival to hypoxic cells. Another important question in this regard is the potential importance of cancer stem cells and whether they may preferentially reside in hypoxic regions of tumours.

Recent advances in imaging technologies suggest that it might be possible within a few years to track, in a non-invasive manner, temporal changes in O2 level and acute and chronic hypoxic tumour subfractions in response to therapy. The ultimate goal will be to under-stand tumour biology in patients on the basis of clinical biomarkers reflecting hypoxia-associated genetic or proteomic signatures in tumour cells in order to assign patients to the best therapy.

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AcknowledgementsThese studies are supported by National Cancer Institute of Canada with funds raised by the Terry Fox Run. R.G.B. is a Canadian Cancer Society Research Scientist.

databasesEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene53BP1 | γH2AX | ARNT | ATM | ATR | BLM | BRCA1 | BRCA2 | CA9 | CAT | CDC25A | CDK2 | CHK1 | CHK2 | CXCR4 | Cyclin E | EIF2α | EIF2AK3 | EME1 | EPAS1 | ERCC1 | FANCD2 | HIF1α | HPRT1 | IL8 | LOX | MAD1L1 | MAX | MDC1 | MDM2 | MLH1 | MNT | MRE11 | MSH2 | MYC | nibrin | p53 | PARP1 | PLAUR | PRKDC | RAD50 | RAD51 | SLC2A | SPP1 | VHL | WAF1 | XRCC5 | XRCC6National Cancer Institute: http://www.cancer.gov/breast cancer | cancer of musculoskeletal tissues | cervical cancer | head and neck cancer | prostate cancerNational Cancer Institute Drug Dictionary: http://www.cancer.gov/drugdictionary/18F-EF5 | azathioprine | cisplatin | etoposide | flavopiridol | methotrexate | misonidazole | mitomycin C | PR-104 | temozolomide | tirapazamine | UCN-01

FURtheR InFoRmatIonRichard P. Hill’s homepage: http://medbio.utoronto.ca/faculty/hill.htmlRobert G. Bristow’s homepage: http://www.radiationatpmh.com/body.php?id=165Spatio-Temporal Targeting and Amplification of Radiation Response: http://www.sttarr.ca/Tumour Microenvironment Group: http://www.uhnres.utoronto.ca/labs/hill/

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