British Journal of Radiology (2006) 79, 545-549
© 2006 British Institute of Radiology
doi: 10.1259/bjr/13634453
Hypoxia in biology and medicine: the legacy of L H Gray
P P Dendy, PhD
1 and
P Wardman, DSc
2
1 1A Coppice Avenue, Great Shelford, Cambridge CB2 5AQ, 2 Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, UK
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Introduction
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On 10 November 2005, the centenary of the birth of Louis Harold Gray FRS (1905–1965), a meeting was held at Blenheim Palace near Oxford to mark the anniversary and to bring together scientists who had worked with Gray and others specializing in the field of tumour hypoxia – which is arguably his main legacy. This Commentary cannot summarize all the presentations, but aims to provide, in this context, a brief overview of the current understanding of hypoxia in relation to radiotherapy and other wider biological and medical implications. We illustrate how progress in imaging hypoxia and understanding changes in gene expression linked to hypoxia provide important avenues towards molecular targeting in radiation oncology, and should eventually lead to individualization of patient treatment to an extent that Gray could only have dreamed of.
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The scientific career of L H Gray
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Barry Michael opened the meeting by outlining Gray's scientific career. "Hal", as he was known to colleagues, studied physics under Rutherford at the Cavendish Laboratory, Cambridge. His mentors at Cambridge included J J Thompson, Chadwick, Cockcroft, C T R Wilson, Aston and Kapitza; a veritable "who's who" of physicists of the early 1930s. At an early stage, Gray decided he wished to use his knowledge in a practical way and turned to biology and medicine. He was elected President of the British Institute of Radiology for 1949–1950 and in 1952 he delivered the 32nd Silvanus Thomson Memorial Lecture, entitled The initiation and development of cellular damage by ionising radiations [1]. The link between tumour blood supply, oxygen delivery and radiosensitivity had been discussed by Mottram in this Journal as early as 1936 [2], but in 1953 and 1955 Gray and his colleagues published two seminal papers [3, 4], recognizing that it might be possible to manipulate the oxygen status to improve radiotherapy. Thus the first paper [3] stated: "Consideration is given to the supply of oxygen to tissues as a factor in radiotherapy, and it is concluded, on the basis of existing knowledge, that in certain circumstances the effectiveness of X-ray treatment might be increased if the patient were breathing oxygen at the time of irradiation." In the later paper [4], Tomlinson and Gray showed that the histological pattern of necrosis in sections of some human lung tumours was consistent with that expected if the supply of oxygen was the limiting factor determining the onset of necrosis. For the remainder of his life, Gray led an enthusiastic team establishing radiobiology as a new, rigorous, scientific discipline. They worked in many areas, but the challenge of hypoxic cells always played a central role and, to this day, remains an increasingly complex problem.
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Hypoxia and radiotherapy: the position in the late 1990s
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The Fifth Edition of Eric Hall's monograph [5] summarizes the state of knowledge on hypoxic cells and hypoxic cell radiosensitizers in the late 1990s. Some key points are:
"Oxygen "fixes" (i.e. makes permanent) the damage produced by free radicals. In the absence of oxygen, damage produced by indirect action may be repaired. Chronic hypoxia results from the limited diffusion range of oxygen through respiring tissue. Acute hypoxia is a result of the temporary closing of tumour blood vessels and is therefore transient. ... There is good evidence that human tumours contain hypoxic cells ... evidence includes histologic appearance, oxygen probe measurements, the binding of radioactive nitroimidazoles ... There is clinical evidence that hypoxia may play an important role in malignant progression."
Hall also summarizes progress on radiosensitizing hypoxic cells, and work towards hypoxia-selective cytotoxins such as tirapazamine. He describes how "Adams and his colleagues listed properties that would be essential for a clinically useful hypoxic cell sensitizer. Firstly, it had to selectively sensitize hypoxic cells at a concentration that would result in acceptable toxicity in normal tissues ... be chemically stable and not subject to rapid metabolic breakdown ... highly soluble in water or lipids ... capable of diffusing a considerable distance through a non-vascularized cell mass to reach hypoxic cells ... effective at ... doses of a few grays."
Several drugs of the nitroimidazole family had been tested as radiosensitizers. Misonidazole had a higher electron affinity and was more effective than metronidazole, being very effective in cells in culture and with animal tumours but with poor results in clinical trials. Related compounds evaluated included etanidazole and nimorazole, which had less toxicity because of shorter biological half-lives or reduced passage across the blood–brain barrier. Nimorazole was shown to be of benefit in head-and-neck cancer in Danish trials, consistent with an earlier meta-analysis by the Danish group of all randomized trials of hypoxia modification indicating an odds ratio of 1.3 [6]. A conclusion from these studies has been that the clinical trials of hypoxic cell radiosensitizers were compromised by a lack of knowledge of the hypoxic status of an individual patient's tumour. Now, however, newer "bioreductive" drugs selectively toxic to hypoxic cells without radiation have been identified. Currently, tirapazamine is the lead compound in clinical trial. Interestingly, these bioreductive drugs also have a dependency on electron affinity for their activity.
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Changes in gene expression
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In recent years, hypoxia-induced changes in gene expression have been extensively demonstrated; two speakers (Bradly Wouters and Christopher Pugh) discussed recent developments. Hypoxia inducible factor (HIF)-1 is a heterodimeric transcription factor made up of 
and 
subunits and it was first recognized as the DNA binding factor that mediates the hypoxia-induced expression of the erythropoietin gene. HIF-1 may promote either directly or indirectly the expression of as many as 60 target genes. Hydroxylation of HIF-1 to an inactive form has an absolute requirement for molecular oxygen and prolyl hydroxylase enzymes: HIF-1 escapes inactivation in hypoxia and hence can bind HIF-1, form the functional HIF-1 complex and drive gene expression. Hypoxia-inducible genes are known to be involved in regulation of biological processes associated with malignancy [7]. HIF-1 can regulate expression of many enzymes in the glycolytic pathway, as well as processes involved in genetic instability, tissue invasion and metastases. HIF-1 also has a clear role in the regulation of genes involved in angiogenesis, both in normal development and in tumours [8]. In all the above roles HIF-1 is likely to promote tumour growth but there are a few situations where HIF-1 (and other isoforms of HIF) can have a negative regulatory effect on tumour response.
The two speakers also discussed different genetically-related issues. Bradly Wouters pointed out that other mechanisms are required to explain the biological response to acute, rather than chronic, hypoxia. He presented new evidence that a potentially important point for regulating gene expression that is able to respond rapidly to changes in the microenvironment is the process of RNA translation (protein synthesis). Global mRNA translation is severely, but reversibly, inhibited during hypoxic conditions, but Wouters and colleagues have shown [9] that, in HeLa cells and prostate carcinoma cells in vitro, this averaging process obscures wide variations in behaviour at the level of individual genes. Indeed a significant number of mRNA species are not dependent on the translation factors that are inhibited during hypoxia and in this efficiently translated fraction of mRNA, 120 genes were more than 4-fold up-regulated by hypoxia.
Pugh pointed out that although HIF is frequently up-regulated in cancer, genetic studies have not always supported a simple model in which up-regulation of HIF promotes a specific biological process associated with malignancy, e.g. angiogenesis, and hence tumour growth [8]. He postulated that genetic mutation may affect the function of an extensive physiological pathway. Thus clonal selection of a particular property affects a package of properties which, individually, could contribute positively, negatively, or not at all to the overall advantage driving selection of the clone. For example, cellular proliferation, HIF activation and angiogenesis might be co-selected because they were linked by pathways that operate physiologically to preserve oxygen homeostasis. Pugh concluded that understanding the HIF system has many implications for cancer biology and interference with this system may have therapeutic uses. However, we will need to know a lot more about the consequences of intervention at any particular point in the development of malignancy to be certain of a net positive benefit.
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Labelling and mapping hypoxic cells
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There have been important developments in markers of hypoxia, especially markers detectable by immunohistochemistry. These markers require no additional intervention beyond an initial pre-treatment biopsy, which is used to generate formalin-fixed or frozen sections, and may be very suitable for widespread clinical use. These markers are more applicable than oxygen electrodes and provide a high resolution assay of the distribution of hypoxia at the microregional level.
Albert van der Kogel discussed imaging the dynamics of tumour hypoxia using immunohistochemical markers [10]. Pimonidazole was injected before biopsy into patients with head and neck cancer, and this can be regarded as an exogenous marker of hypoxia. Following sectioning, carbonic anhydrase CA9 (a HIF-dependent gene that has been investigated as a putative endogenous hypoxia marker) and blood vessels could also be visualized by immunohistochemistry. Beautiful triple-staining images of pimonidazole, CA9 and vessels were shown. Kaanders et al [10] confirmed the widespread variation in hypoxia in human tumours of the head and neck – in 43 squamous cell carcinomas, tumour area staining positive for pimonidazole ranged from 0.3% to 17.2%. Follow up of patients after treatment showed a significant correlation between vascular density and locoregional control, and a significant negative correlation between pimonidazole binding and both locoregional control (15 months) and disease-free survival (2 years).
In the laboratory, more sophisticated experiments are possible because of the use of experimental models. For example, van Laarhaven et al [11] injected two nitroimidazole markers of hypoxia, CCI-103F and pimonidazole, before and after treatment with nicotinamide and carbogen (95% O2, 5% CO2) singly and in combination into two different murine carcinomas. Bromodeoxyuridine and Hoechst 33342 were also used as proliferation and perfusion markers, respectively. The two tumours showed extensive differences in vascular architecture, distribution patterns of hypoxia and bromodeoxyuridine labelling. All treatment combinations caused a decrease in the hypoxic fraction, but the responses of the two tumours were quantitatively very different.
As an alternative to image analysis of histological sections, flow cytometry was used by Bennewith and Durand [12] to obtain important information about the transient nature of hypoxia. Pimonidazole was injected hourly into a human tumour xenograft for 8 h. This causes a time-integrated asymptotic rise in the number of cells showing the hypoxic marker. 1 h before sacrifice, a second hypoxic marker (CCI-103F) was injected. Examination by flow cytometry of the single and dual fluorescence peaks from a large number of cells showed that substantial numbers of cells that had been hypoxic were no longer hypoxic immediately before sacrifice. This dynamic behaviour is likely to have a big influence on tumour management and particularly the application of hypoxia-directed therapies.
Methods of monitoring hypoxia using non-invasive imaging techniques are being explored. Nitroimidazoles labelled with I-123 may be suitable for conventional gamma camera imaging or with F-18 for PET imaging. A recent paper by Nöth et al [13] described the use of 15C5-loaded alginate capsules as fluorine-19 oxygen sensors in MRI for in vivo determination of tumour oxygenation during growth and in response to carbogen breathing.
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Molecular targeting in radiation oncology
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Addressing this subject, Michael Baumann pointed out that radiotherapy is very efficient at reducing a tumour to a small number of surviving clonogenic cells. There is therefore scope for developing novel therapeutic agents which, although perhaps not curative in themselves, may be highly effective if used in combination with an appropriate therapeutic regimen. Hypoxia is known to have a role in: (i) selecting for cells that have lost sensitivity to the tumour suppressor gene p53; (ii) regulating genes involved in drug resistance; (iii) a tendency to select for a more malignant phenotype; (iv) increasing the mutation rate; (v) increasing expression of genes associated with angiogenesis; and (vi) tumour invasion. There is therefore considerable potential for developing new approaches to therapy based on targeting hypoxia. Two examples discussed by Baumann are summarized below.
A number of groups have shown a link between expression of epidermal growth factor receptor (EGFR) and tumour growth. For example, Ang et al [14] showed that in a large series of patients with head and neck squamous cell carcinomas, EGFR expression was a strong independent prognostic indicator of overall and disease-free survival. It was also a robust predictor of locoregional relapse, but not of distant metastases. The authors recommended that EGFR immunohistochemistry should be considered for selecting patients for more aggressive combined therapies or enrolment in trials targeting EGFR signalling pathways. Baumann and Krause [15] had reviewed the evidence that inhibition of EGFR can increase radiosensitivity of clonogenic cells and tumour cell proliferation, and Krause et al [16] have shown that in nude mice the anti-EGFR monoclonal antibody can significantly reduce the tumour control dose with decreased repopulation and reoxygenation.
The second example targets tumour angiogenesis. Adjuvant inhibition of the vascular endothelial growth factor receptor (VEGFR) with a tyrosine kinase inhibitor after fractionated irradiation prolongs tumour growth [17]. Zips et al [18] have confirmed this finding for a human squamous cell carcinoma grown in nude mice and exposed to the VEGFR inhibitor for 75 days after irradiation, but showed that the 50% tumour control dose was no different. The authors conclude that recurrences depend on VFGF-driven angiogenesis but surviving tumour cells retain their clonogenic potential during this treatment.
Other strategies for targeting therapies based on hypoxia, not discussed in detail at the meeting, include hypoxia-activated prodrugs, hypoxia-selected gene therapy, and the use of genetically-engineered anaerobic bacteria [19]. Molecular-based approaches targeted to hypoxia-mediated processes add a further dimension to the complexity of the problem since overall and disease-free survival, locoregional control, and control of metastases rarely give concordant results in animal experiments and clinical trials.
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Clinical trials
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Van der Kogel also outlined the background to the present Phase 3 clinical trials of ARCON (accelerated radiotherapy with carbogen and nicotinamide) in head and neck and bladder cancers [20]. This concept, which was pioneered at the Gray Laboratory, is designed to attack tumour cells that may have more than one resistance mechanism (i.e. acute and chronic hypoxia) and therefore may be responsive to combined modality treatments. Furthermore, tumour repopulation is a major cause of poor local tumour control, which generally deteriorates as treatment time increases (at fixed dose). Thus the first arm of the ARCON attack is to use accelerated fractionated radiotherapy with several fractions per day. Inhalation of carbogen is designed to decrease diffusion-limited hypoxia and nicotinamide is used to decrease perfusion-limited hypoxia, although it may also have other effects.
ARCON is a good example of translational research. Pre-clinical studies, mainly on animals, have shown that each of the three components can be effective, both in isolation and in combination. For example, tumour control rate for a mouse mammary carcinoma was the same with the combination treatment with almost 50% less radiation dose. Phase 1 and 2 clinical trials showed that the ARCON regimen was feasible and tolerable in patients and produced promising results in terms of tumour control. For the Phase 3 trial that is nearing completion, selection of patients has been mainly based on clinical and histopathological tumour characteristics. It is already clear that, whatever the outcome, improved selection of patients based on measures of hypoxia will be desirable, if not essential, to achieve good therapeutic outcomes, as may now be appreciated from previous hypoxic cell radiosensitizer trials.
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Widening horizons
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Two speakers took a broader view of some aspects of radiobiology. Using the title Science spanning the generations, Ian Stratford reviewed a number of other areas in which early work by Gray was taken forward by colleagues and later generations with varying degrees of success. One phenomenon that attracted the attention of Gray and co-workers was the effect of nitric oxide on the radiosensitivity of tumour cells [21, 22]. Nitric oxide appeared to be able to take the place of oxygen in enhancing sensitivity to X-rays in bacteria, plant meristem and mammalian tumour cells. Furthermore, cell survival curves showed that sensitization by nitric oxide was exactly the same as that achieved with oxygen when added to anoxic conditions. It is now known that nitric oxide is a biochemical signalling molecule, generated in tissues by nitric oxide synthase (NOS). NOS levels are elevated in a wide range of tumour types (see, for example, Ambs et al [23]). Stratford showed that cytokine-mediated induction of NOS radiosensitized tumour cells at intermediate oxygen tensions in vitro, hence suggesting that varying levels of NOS in human tumours could be a significant factor in radiotherapy.
Another topic studied by Gray was the dissociation of oxyhaemoglobin [24]; Adams, Stratford and colleagues subsequently explored enhancement of hypoxia by compounds such as BW12C which influenced this dissociation. They showed that these compounds could increase tumour hypoxia and thereby be useful when combined with hypoxia-selective bioreductive drugs [25]. An alternative approach has been taken in the development of efaproxiral (RSR13), which reduces haemoglobin–oxygen binding to decrease hypoxia [26]; this agent is now in clinical trial in combination with radiotherapy.
Stratford also discussed the extensive research on the development of bioreductive drugs worldwide, some pioneered at the Gray Laboratory [19, 25, 27], and the potential for utilizing the presence of hypoxia in tumours to drive selective gene therapy [28]. Finally, he returned to the impact of modulating HIF function on tumour response to radiotherapy, presenting data to support the hypothesis that in HIF-1 deficient tumours, hypoxic cells have an extremely limited lifetime and therefore may not contribute to radiation sensitivity [29].
Gillies McKenna outlined the research programme of the newly-established Department of Radiation Oncology and Biology at the University of Oxford, which will incorporate the Gray Laboratory when it is moved from its current location at Mount Vernon Hospital. The vision of the Department is to continue the tradition of the "Gray Lab"; be research focused and clinically-relevant; learn lessons from the past; be flexible to encompass all relevant areas in the future; complement existing groups in Oxford and elsewhere in the UK; and be a centre for training all health care personnel in radiation oncology and biology. Multidisciplinary collaboration will be a key feature of the Department, and two examples were given.
First, in the 21st century it will be necessary to move on from empirical therapy, tailored by experience, to targeted therapy. Maximizing the information from tumour imaging will involve diagnostic radiologists, medical physicists, radiographers, and others. Pushing back the boundaries of physical treatment involves radiotherapists, radiation physicists and industry. To these groups must be added a broad spectrum of biologists who will open up new frontiers. McKenna and his colleagues are seeking to define biological processes that regulate the radiation responsiveness of solid tumours, and take agents that target these processes into the clinic in order to improve therapeutic outcome.
Second, there will be strong emphasis on translational research. Examples of areas where laboratory workers and clinicians must work in close collaboration include signal transduction inhibitors with radiation, systematic targeting of radiation with Auger electron-emitting radionuclides, molecular targets for modifying the radiation response, and the molecular basis of metastases.
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Conclusions
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Hal Gray was a true polymath: introducing their biography of him, Loutit and Scott discussed Gray's "escape from this confinement" of the "cult of specialization for learning more and more about less and less" [30]. One of us (PW), writing in 1982 in a booklet to commemorate the silver jubilee of the opening of the Gray Laboratory, asserted "... [Gray] must have been the first – and quite possibly the last – scientist to have a thorough appreciation of current activity in all four sectors of radiation research – physics, chemistry, biology and medicine." While no one subject can sum up his work as a scientist, overcoming tumour hypoxia is a challenge that requires such a broad approach. Had he not died so young he might well have received a Nobel Prize. Some of his contributions are now well understood, for example radiation dosimetry, where Gray's name is recorded for posterity as the SI unit of absorbed dose. Other areas, especially hypoxia in tumours, represent a far more complex problem than Gray could possibly have imagined, with heterogeneity of behaviour in almost every aspect investigated.
In future, human tumours must be better characterized on an individual basis. Performing clinical trials on unselected patients who have a mixture of hypoxic and better-oxygenated tumour cells runs the clear risk of rejecting a treatment that could be of significant benefit to a sub-set of patients. Loutit and Scott described Gray as "the Fellow [of The Royal Society] who fathered radiobiology" [30], but it is arguable that drawing attention to the importance of tumour hypoxia is the most important legacy of L H Gray.
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Acknowledgments
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We thank Prof. I J Stratford for helpful comments on a draft manuscript. PW is supported by Cancer Research UK.
Received for publication March 15, 2006.
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References
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Introduction
The scientific career of...
