Australas Radio1 1992: 36: 85-89
Conference Report: 9th International Congress of Radiation Research Toronto, Canada, July 7-12,1991 MICHAEL J. McKAY, F.R.A.C.R. National Health and Medical Research Council Postgraduate Research Scholar Department of Medical Oncology, Westmead Hospital, Westmead 2145, NSW. MICHAEL BARTON, F.R.A.C.R. Consultant Radiation Oncologist, Institute of Oncology Department of Radiotherapy The Prince of Wales Hospiral, Randwick, NSW
INTRODUCTION An International Congress of Radiation Research con-
venes every four years. The 9th such congress was held at the Sheraton Centre, Toronto, Canada, between the 7th and 12th of July, 1991. Major congress sponsors were the Radiation Reseach Society, The International Association of Radiation Research and the North American Hyperthermia Group. This particular meeting was of a high scientific quality, bringing together many experts in those research disciplines relating to radiation. Although the emphasis was on ionising radiation, other areas were covered, including ultraviolet radiation (UV) and ultrasound. This report focuses on ionising radiation, except where reference to other radiations helps illustrate a particular point. The aim of this report is to highlight some of the major areas of current interest in radiation research. Because references cannot be included, the potential risks of misquoting dicate that only general concepts be presented. The report is also selective, in that only those sessions attended by the authors are covered. The text represents a mixed bag of information arising from lectures, symposia, workshops and poster sessions, as well as the notes taken in these sessions, and the authors accept responsibility for any errors of reporting or omission. In introducing a number of topics, a brief statement or two summarising the present state of knowledge, is given. Notwithstanding the above caveats, it was felt that the meeting provided sufficient important innovations in both basic and clinical research to justify this communication. It is hoped that it will be of some interest not only to radiation oncologists,but to diagnostic radiologists as well.
1. Some aspects of X-ray induced biological damage There is good evidence that damage to cellular DNA is a fundamental event in X-ray induced cell death. Although DNA double strand breaks are thought to be qualitatively the most important DNA lesion, X-rays also Key words: Radiation, research, conference report Address for correspondence: Michael J. McKay National Health and Medical Research Council Postgraduate Research Scholar Department of Medical Oncology Weshnead Hospital Westmead 2145 NSW
Australasian Radiology, Vol.36, No. I , February, I992
cause other lesions, such as single strand breaks and modification or loss of bases. Eric Hall (Columbia University, New York, USA) chaired a debate entitled: ‘DNA doublestrand breaks are the only lesions that kill mammalian cells exposed to ionising radiation’. The four panel members argued initially for, and then against, the motion. The outcome of the debate was decided by an audience vote, the majority deciding against the motion. It was recognised that other lesions, for example DNA base damage, can by themselves cause cell death, but that their quantitative importance remains to be elucidated. Gordon Steele (Institute of Cancer Research, Surrey, UK) emphasised the concept of the locally multiply damaged site (‘LMDS’) as the critical biological lesion induced by X-rays. These lesions resulted from ionisation clusters, and are characteristic of both high and low LET (linear energy transfer) radiation. He indicated that the LMDS, although most often associated with a DNA double strand break, was not simply a ‘clean cut’ but a complex mixture of a number of different types of lesion. Because of the complex nature of such lesions, choosing the appropriate type of damage to study was difficult. It follows that standard in vitro assays for repair after X-ray damage (for example, the rate and fidelity of DNA double strand break repair) should only be considered an approximation of the ability of the cells in question to repair X-ray damage. That the microstructure of DNA strand breaks is critical was also addressed by Tomas Lindahl (Clare Hall Laboratories, Herts, UK). His group continues to design cell-free assays to evaluate the repair of one specific DNA lesion at a time. This approach is hoped to allow delineation of the most important X-ray induced DNA alterations at the molecular level, as well as the enzymes involved in their repair. The approach has already yieIded success in identifying a deficiency in the DNA ligase-I enzyme as the major defect in the cancer-prone hereditary disorder, Bloom’s syndrome. In discussing X-ray induced mutagenesis, John Thacker (Oxford University, Chilton, Didcot, UK), indicated that ionising radiations cause large DNA lesions, for example, chromosomal rearrangements and large deletions. This is in contrast to alkylating agents, for example, which typically result in point mutations. Not only do different genotoxic agents result in a different mutational Submitted for publication on: 20th December, 1991 Accepted for publication on: 23rd December, 1991
85
M.J. McKAY AND M. BARTON
spectrum, but the experimental system used to evaluate the mutations can give quite different results. He quoted markedly different mutation frequencies at different gene loci in hamster cells, after exposure to X-rays and other agents. These results indicated that lesion induction or lesion processing can be different in different parts of the genome. On a similar theme, Franklin Hutchinson (Yale University, New Haven, USA) reported that when the bacterium E. coli was x-irradiated, suprisingly, no deletions or rearrangements were seen in the resulting mutants. One postulated reason for this was that in contrast to the mammalian genome, E.co1i has only very short stretches of non-coding DNA between functional parts of genes, and as a result, any large X-ray induced lesion would be expected to inactivate relatively more essential stretches of DNA (including a large number of genes and such structures as DNA replication origins), and hence be lethal. Thus, this type of mutation was not detected. This contribution again emphasised the importance of recognising the limitations of any one of the presently available experimental systems for evaluating X-ray induced mutations. M. Comforth (Los Alamos National Laboratory, Los Aiamos, USA), stated that the majority of X-ray induced chromosomal aberrations are exchange aberrations, with only a small percentage (eg. 3-5%), being deletions. He indicated that the majority of studies had shown that these exchanges demonstrated no particular site predilection, although Joel Bedford (Colorado State University, Fort Collins, USA) reported a new experimental system where the contrary was found. In this system, a highly amplified plasmid (an independently replicating DNA unit) had been inserted into mosquito cells, and functioned as a stably integrated extra chromosome. This plasmid was shown to be approximately twice as susceptible to aberration induction as the host genetic material. Attempts to determine the biologic basis for this enhanced susceptibility were proceeding. It has for some time been recognised that the densely packed nuclear chromatin (heterochromatin), in particular that located adjacent to the nuclear membrane, is more susceptible to DNA damage from X-rays. A number of presentations addressed this concept. One possible explanation for the effect was offered by Phillip Hanawalt (Stanford University, Stanford, USA). An open chromatin pattern is associated with a higher proportion of actively transcribing genes. There is now direct evidence that damage to these regions affords a greater accessibility to DNA repair enzymes, almost certainly explaining the above effect.
2. DNA repair-deficient diseases The ‘other side of the coin’ to DNA damage is DNA repair. There are a number of human disorders charactensed by defects in repair of DNA damage induced by either UV or ionising radiation. Most such disorders are associated with an increased risk of cancer. K. Kraemer (National Cancer Institute, Maryland, USA) discussed 9 such disorders (Table). Ataxia telangiectasia (AT) is the prototypical human disorder characterised by an enhanced sensitivity to ionising radiation. This sensitivity is manifest not only at a clinical level, but also in vitro. To date, there have been 4 different cellular subgroups (so called complementation groups) identified. These cellular subgroups however, are indistinguishable at the clinical level. Each subgroup is presumably representative of a defect in
86
a particular gene. In fact, 3 genes for this disorder have recently been genetically mapped to chromosome 11. A variant of AT, the Nijmegen breakage syndrome, as well as Gorlin’s (basal cell naevus) syndrome, comprise the remaining human syndromes showing clinical X-ray hypersensitivity. Interestingly, fibroblasts from individuals with Gorlin’s syndrome demonstrate normal radiosensitivity in vitro. The remaining disorders listed in the table manifest UV hypersensitivity.
Human DNA repair deficiency disorders 1. Ataxia telangiectasia 2. Nijmegen breakage syndrome 3. Gorlin’s syndrome 4. Xeroderma pigmentosum
5. Cockayne’s syiidrome’
6. Fanconi’s anaemia 7. Bloom’s syndrome 8. Trichothyrodystrophy*
9. Dysplastic naevus syndrome TABLE. The human DNA repair deficiency states. There is an enhanced risk of malignancy in these conditions, except those marked with an asterisk. The first three conditions demonstrate X-rav hypersensitivity, the remainder, UV hypersensitivity. Cellular hypersensftivity to UV is not seen in all cell-based studies in dysplastic naevus syndrome.
3. DNA repair and DNA repair genes After exposure to a wide variety of DNA-damaging agents, as part of a cellular survival response, many genes become transcriptionally active (ie. synthesise messenger RNA). The majority of these genes are not directly involved in repairing the damaged DNA, however in the last few years a number of human repair genes have been identified, most of which are involved the the repair of damage caused by UV radiation. Largely because of the complexity of X-ray induced damaged, there has to date been only one such X-ray repair gene identified, XRCC- 1 (X-ray Repair Cross Complementing Chinese hamster ovary cells). Although the function of XRCC-1 is largely unknown, it is known to be associated with the repair of single strand DNA breaks. New strategies to further study the biologic function of XRCC-1 include the construction of transgenic mice, deficient in the function of this gene (Larry Thompson; Lawrence Livermore National Laboratory, Livermore, USA). The biological relevance of the cloned repair genes is graphically illustrated by those human disorders resulting from the lack of function of a single one of these genes. For example, Christine Weber (Lawrence Livermore National Laboratory, Livermore, USA) reported that her group have recently demonstrated that a UV repair gene, ERCC-2 (Excision Repair Cross Complementing Chinese hamster ovary cells), fully corrects the repair defect in cell lines from one subset of xeroderma pigmentosum (XP) individuals [ie. ERCC-2 is the gene deficient in this condition (XP-D)]. Previously, groups from Japan and the Netherlands had shown that genetic complementation of other XP subsets was possible, using two of the other five cloned ERCC genes. All six ERCC genes have been isolated using rodent systems. Australasian Radiology. VoL 36. No. I , Fehruai?. 1992
9TH INTERNATIONAL CONGRESS OF RADIATION RESEARCH, TORONTO, CANADA, 7-12 JULY 1991
These studies thus underscore the contribution of such laboratory models to an understanding of human genetic diseases. Dirk Bootsma (Erasmus University, Rotterdam, The Netherlands), gave an overview of the present state of knowledge regarding the six ERCC genes. All six genes encode proteins involved in the incision step of UV excision (‘patch’) repair. Despite this, the genes vary significantly in size, and code for protein sequences which would, on the basis of structure alone, be predicted to have diverse biological roles. As was indicated earlier, Phillip Hanawalt (Stanford University, Stanford, USA) stressed that, at least as far as UV radiation was concerned, actively transcribing genes demonstrate preferential repair. This pattern has held true for every human locus examined to date. No such data is presently available for ionising radiation. More recently, it was shown that even within preferentially repaired genes, lesions on the DNA strand which was actively transcribing ( t o mRNA), were more efficiently removed. Futhermore, there is evidence that UV-induced damage to inactive genes in the immediate vicinity of a preferentially repaired, active gene, can enjoy augmentation of repair, implicating a regional repair effect. The basic experimental substrate for the isolation of human DNA repair genes have been rodent cell ‘mutants’, deficient in one or more aspects of DNA repair. In the case of the cloned repair genes, the rodent cell deficiency was due to absence or inactivation of that particular gene. The addition of human DNA complemented the rodent defect. This allowed the respective genes to be identified. Over 20 such X-ray repair-deficient rodent strains have been isolated and characterised by multiple laboratories, since 1983. Three more such variants were described, by Murray Stackhouse (Colorado State University, Fort Collins, USA), A. Varghese (Ontario Cancer Institute, Toronto, Canada) and by a Sydney University group (MM). As an alternative approach to the study of genetic factors governing radiosensitivity, Jack Russell (Glasgow, UK) reported the isolation of a radioresistant neuroblastoma cellular variant from the repeated exposure of a radiosensitive parent line to X-irradiation. It is possible that such a variant may demonstrate, for example, amplification of genes involved in DNA repair. Discussing the enzymology of DNA repair in E. coli, Susan Wallace (University of Vermont, Vermont, USA) (President Elect of the Radiation Research Society) emphasised that DNA base excision repair enzymes, responsible for repair of qualitatively minor X-ray induced lesions, were ubiquitous, highly conserved proteins. Identification and molecular cloning of such bacterial genes was thus relevant to the isolation of their human homologues (equivalents). Three such genes have been cloned to date.
4. Molecular genetics of tumour progression Webster Cavanee (Montreal University, Montreal, Canada) presented compelling molecular evidence for the biological progression of low grade, through intermediate grade, to high grade cerebral astrocytomas. For example, in over 120 glioblastomas multiforme examined, over 95% had a constant abnormality associated with chromosome 10 (a deletion), found in none of the lower grade tumours. All grades of astrocytoma examined were associated with another fixed abnormality of chromosome 17 (also a deletion). Epidermal growth factor receptor (EGFR) was amplified preferentially in intermediate, and Australasian Radiology Vol.36. No. I. Fehruap, 1992
to a lesser extent, high grade tumours. These facts alone should facilitate more accurate diagnosis of these tumours. Perhaps more importantly, they strongly suggest an ordered succession of genetic abnormalities during a progression from low to high grade cancers, confirming an observation first made in relation to colorectal cancer. As such, gliomas may also prove a useful model for tumour progession at other sites. A further point was that the degree of aggressiveness of a number of glioma cell lines (as measured by invasion into foetal mouse brain) was reduced by successful transfection (transfer) of the missing chromosomal material into the glioma line, indicating the possible therapeutic application of the identification of deleted/inactivated tumour supressor genes (TSG’s) in cancers of different sites.
5. X-ray induced gene expression There are described an emerging number of genes, which are inducible by X-rays and other DNA damaging agents, as discussed by A1 Fornace (National Cancer Institute, Maryland, USA). In relation to X-rays, one general group is activated via a protein kinase C (PKC) pathway. These genes include the well characterised ‘immediate early’ genes: eg. jun, fos, E G R l , tumour necrosis factor, and a group of genes involved in negative growth control (eg the gadd-Growth Arrest and DNA Damage inducible-genes). In general, the study and characterisation of these sorts of genes may lead to the identification of those which are associated with intrinsic cellular radioresistance, however, to date there have been major difficulties distinguishing the genes of interest from the high background of induced genes not directly participating in DNA repair.
6. Oncogene activation during X-ray induced cellular transformation It is well established, both in the laboratory and clinic, that X-rays can induce the development of malignant cellular characteristics in benign cells. Analagous to the situation demonstrated at a number of other sites [notably the large bowel and more recently, for cerebral gliomas (see section 4)] the mechanism of this effect presumably involves a mutistep activation of dominantly acting cellular protooncogenes, in association with the inactivation of growth inhibitory (‘tumour supressor’) genes. The exact sequence of mutational events, however, remains to be shown, and may be different for different tissues. Using mouse 3T3 cells, Jack Little (Harvard University, Boston, USA) and colleagues examined cell colonies transformed after X-rays (6Gy), for evidence of activation of oncogenes, using Southern analysis, as well as a more sensitive technique (denaturing gel gradient electrophoresis). All 15 dominantly-acting oncogenes examined, including the Ras family of proteins, showed no induction when compared with controls. The p53 protein, which has both dominant and recessive (tumour supressive) effects, was mutated in approximately 20% of the clones examined. Further attempts to elucidate molecular concomitants of neoplastic transformation by X-rays are proceeding. In summing up, Professor Little hypothesised that because genetic recombination (a normal event at meiosis) was a major repair pathway for DNA double strand breaks (critical X-ray induced lesions), activation of a general recombination machinery by X-rays may account for their substantiaf transforming ability, by altering the normal spatial relationship between oncogenes and their promoters/ enhancers. This could result in altered gene expression. The actual oncogenes implicated require definition. 87
M.J. McKAY AND M. BARTON
7. Cell cycle biology and radiation There has been considerable recent interest in cellular proteins which induce, or are associated with, the various cell cycle phases. Whilst it has been recognised for some time that the G2-M boundary was an important checkpoint, in that, once passed, the cell was committed to mitosis, its molecular basis has until recently, been unclear. Joseph Roti-Roti (St Louis, USA) reported on studies in yeast which have recently identified a number of specific protein kinases which appear to mediate this effect. Radiation sensitive yeast mutants have shown that the lack of a radiation-induced G2-M block greatly reduces the fidelity and completeness of DNA repair after X-irradiation. These yeast proteins show a strong homology to human proteins with the same function. One of the basic cellular defects in cells from individuals with ataxia telangiectasia is an enhanced cellular accumulation at the G2-M boundary after X-irradiation. Whether these proteins or their regulation is abnormal in this condition is presently being evaluated. Reporting on studies evaluating G2 phase delay after ionising radiation, W. Gillies McKenna (University of Pennsylvania, Pennsylvania, USA) pointed out that the appearance of the mRNA for a specific protein kinase, cyclin B, was associated with the end of the normal G2 delay in mammalian cells. He postulated an addition to the list of possible contributors to intrinsic cellular radioresistance, namely, inhibitors of, or defects in, cyclin €3 expression, which may thereby allow greater damage processing before a cell enters mitosis.
8. Proton beams in radiation therapy I n the Ontario Cancer Treatment and Research Foundation (Richards) Award lecture, Herman Suit (Massachusetts, USA) provided an overview of the experience of the Massachusetts General Hospital and its collaborators with proton beam therapy since 1972. Although the clinical spectrum of diseases for which this modality is useful or appropriate is limited, impressive results have accrued in the treatment of uveal melanomas and sarcomas of the skull base. In the case of the former, over 1600 patients have been treated, with 5 year local control within the high dose volume of 99% (and an overall local control of 96%), and 5 year survival of approximately 80%. Preservation of vision in the vast majority of patients is the obvious advantage over surgery. These figures are mirrored in the other two functional proton facilities (one US, one French, having treated approximately 350 and 900 patients respectively). 9. Assays predictive of radiation response There is great interest in developing clinically relevant assays of tumour (and normal tissue) radiosensitivity. Such predictive assays may help define which subsets of tumours may be better treated with altered therapies such as concurrent chemotherapy or accelerated fractionation. For example, tumours with short doubling times may be better treated with the latter approach.
a. In vitro clonogenic cell survial assays The surviving fraction of cells after 2Gy (SF2) has been shown to correlate in some cases with variations in clinically observed tumour radiosensitivities. This test involves developing a primary tumour cell culture from a biopsy and as such is relatively slow, laborious and costly. On average, 70% of biopsies taken develop into usable cultures. Early data are available on two assays, tha CAM clonogenic assay and the Courtney clonogenic assay.
88
William Brock (MD Anderson Cancer Center, Houston, USA), whose group developed the CAM assay, presented 140 patients with head and neck (H & N) cancer who received post-operative radiation therapy. SF2 ranged from 0.10 to 0.92, with a mean of 0.32. They found no significant correlation between SF2 and local control but their results are difficult to interpret because surgery was the primary treatment modality, T. Girinsky (Institute Gustave-Roussy, Villejuif, France) showed information on 97 H & N and cervix tumours treated primarily by radiation therapy, using the CAM assay to calculate SF2. A cell growth fraction was also calculated by flow cytometry using cells labelled with bromodeoxyuridine. The mean SF2 for cervix and H & N cancers was similar (0.39 and 0.38 respectively). Tumours with SF2
Conference Report: 9th International Congress of Radiation Research Toronto, Canada, July 7-12,1991 MICHAEL J. McKAY, F.R.A.C.R. National Health and Medical Research Council Postgraduate Research Scholar Department of Medical Oncology, Westmead Hospital, Westmead 2145, NSW. MICHAEL BARTON, F.R.A.C.R. Consultant Radiation Oncologist, Institute of Oncology Department of Radiotherapy The Prince of Wales Hospiral, Randwick, NSW
INTRODUCTION An International Congress of Radiation Research con-
venes every four years. The 9th such congress was held at the Sheraton Centre, Toronto, Canada, between the 7th and 12th of July, 1991. Major congress sponsors were the Radiation Reseach Society, The International Association of Radiation Research and the North American Hyperthermia Group. This particular meeting was of a high scientific quality, bringing together many experts in those research disciplines relating to radiation. Although the emphasis was on ionising radiation, other areas were covered, including ultraviolet radiation (UV) and ultrasound. This report focuses on ionising radiation, except where reference to other radiations helps illustrate a particular point. The aim of this report is to highlight some of the major areas of current interest in radiation research. Because references cannot be included, the potential risks of misquoting dicate that only general concepts be presented. The report is also selective, in that only those sessions attended by the authors are covered. The text represents a mixed bag of information arising from lectures, symposia, workshops and poster sessions, as well as the notes taken in these sessions, and the authors accept responsibility for any errors of reporting or omission. In introducing a number of topics, a brief statement or two summarising the present state of knowledge, is given. Notwithstanding the above caveats, it was felt that the meeting provided sufficient important innovations in both basic and clinical research to justify this communication. It is hoped that it will be of some interest not only to radiation oncologists,but to diagnostic radiologists as well.
1. Some aspects of X-ray induced biological damage There is good evidence that damage to cellular DNA is a fundamental event in X-ray induced cell death. Although DNA double strand breaks are thought to be qualitatively the most important DNA lesion, X-rays also Key words: Radiation, research, conference report Address for correspondence: Michael J. McKay National Health and Medical Research Council Postgraduate Research Scholar Department of Medical Oncology Weshnead Hospital Westmead 2145 NSW
Australasian Radiology, Vol.36, No. I , February, I992
cause other lesions, such as single strand breaks and modification or loss of bases. Eric Hall (Columbia University, New York, USA) chaired a debate entitled: ‘DNA doublestrand breaks are the only lesions that kill mammalian cells exposed to ionising radiation’. The four panel members argued initially for, and then against, the motion. The outcome of the debate was decided by an audience vote, the majority deciding against the motion. It was recognised that other lesions, for example DNA base damage, can by themselves cause cell death, but that their quantitative importance remains to be elucidated. Gordon Steele (Institute of Cancer Research, Surrey, UK) emphasised the concept of the locally multiply damaged site (‘LMDS’) as the critical biological lesion induced by X-rays. These lesions resulted from ionisation clusters, and are characteristic of both high and low LET (linear energy transfer) radiation. He indicated that the LMDS, although most often associated with a DNA double strand break, was not simply a ‘clean cut’ but a complex mixture of a number of different types of lesion. Because of the complex nature of such lesions, choosing the appropriate type of damage to study was difficult. It follows that standard in vitro assays for repair after X-ray damage (for example, the rate and fidelity of DNA double strand break repair) should only be considered an approximation of the ability of the cells in question to repair X-ray damage. That the microstructure of DNA strand breaks is critical was also addressed by Tomas Lindahl (Clare Hall Laboratories, Herts, UK). His group continues to design cell-free assays to evaluate the repair of one specific DNA lesion at a time. This approach is hoped to allow delineation of the most important X-ray induced DNA alterations at the molecular level, as well as the enzymes involved in their repair. The approach has already yieIded success in identifying a deficiency in the DNA ligase-I enzyme as the major defect in the cancer-prone hereditary disorder, Bloom’s syndrome. In discussing X-ray induced mutagenesis, John Thacker (Oxford University, Chilton, Didcot, UK), indicated that ionising radiations cause large DNA lesions, for example, chromosomal rearrangements and large deletions. This is in contrast to alkylating agents, for example, which typically result in point mutations. Not only do different genotoxic agents result in a different mutational Submitted for publication on: 20th December, 1991 Accepted for publication on: 23rd December, 1991
85
M.J. McKAY AND M. BARTON
spectrum, but the experimental system used to evaluate the mutations can give quite different results. He quoted markedly different mutation frequencies at different gene loci in hamster cells, after exposure to X-rays and other agents. These results indicated that lesion induction or lesion processing can be different in different parts of the genome. On a similar theme, Franklin Hutchinson (Yale University, New Haven, USA) reported that when the bacterium E. coli was x-irradiated, suprisingly, no deletions or rearrangements were seen in the resulting mutants. One postulated reason for this was that in contrast to the mammalian genome, E.co1i has only very short stretches of non-coding DNA between functional parts of genes, and as a result, any large X-ray induced lesion would be expected to inactivate relatively more essential stretches of DNA (including a large number of genes and such structures as DNA replication origins), and hence be lethal. Thus, this type of mutation was not detected. This contribution again emphasised the importance of recognising the limitations of any one of the presently available experimental systems for evaluating X-ray induced mutations. M. Comforth (Los Alamos National Laboratory, Los Aiamos, USA), stated that the majority of X-ray induced chromosomal aberrations are exchange aberrations, with only a small percentage (eg. 3-5%), being deletions. He indicated that the majority of studies had shown that these exchanges demonstrated no particular site predilection, although Joel Bedford (Colorado State University, Fort Collins, USA) reported a new experimental system where the contrary was found. In this system, a highly amplified plasmid (an independently replicating DNA unit) had been inserted into mosquito cells, and functioned as a stably integrated extra chromosome. This plasmid was shown to be approximately twice as susceptible to aberration induction as the host genetic material. Attempts to determine the biologic basis for this enhanced susceptibility were proceeding. It has for some time been recognised that the densely packed nuclear chromatin (heterochromatin), in particular that located adjacent to the nuclear membrane, is more susceptible to DNA damage from X-rays. A number of presentations addressed this concept. One possible explanation for the effect was offered by Phillip Hanawalt (Stanford University, Stanford, USA). An open chromatin pattern is associated with a higher proportion of actively transcribing genes. There is now direct evidence that damage to these regions affords a greater accessibility to DNA repair enzymes, almost certainly explaining the above effect.
2. DNA repair-deficient diseases The ‘other side of the coin’ to DNA damage is DNA repair. There are a number of human disorders charactensed by defects in repair of DNA damage induced by either UV or ionising radiation. Most such disorders are associated with an increased risk of cancer. K. Kraemer (National Cancer Institute, Maryland, USA) discussed 9 such disorders (Table). Ataxia telangiectasia (AT) is the prototypical human disorder characterised by an enhanced sensitivity to ionising radiation. This sensitivity is manifest not only at a clinical level, but also in vitro. To date, there have been 4 different cellular subgroups (so called complementation groups) identified. These cellular subgroups however, are indistinguishable at the clinical level. Each subgroup is presumably representative of a defect in
86
a particular gene. In fact, 3 genes for this disorder have recently been genetically mapped to chromosome 11. A variant of AT, the Nijmegen breakage syndrome, as well as Gorlin’s (basal cell naevus) syndrome, comprise the remaining human syndromes showing clinical X-ray hypersensitivity. Interestingly, fibroblasts from individuals with Gorlin’s syndrome demonstrate normal radiosensitivity in vitro. The remaining disorders listed in the table manifest UV hypersensitivity.
Human DNA repair deficiency disorders 1. Ataxia telangiectasia 2. Nijmegen breakage syndrome 3. Gorlin’s syndrome 4. Xeroderma pigmentosum
5. Cockayne’s syiidrome’
6. Fanconi’s anaemia 7. Bloom’s syndrome 8. Trichothyrodystrophy*
9. Dysplastic naevus syndrome TABLE. The human DNA repair deficiency states. There is an enhanced risk of malignancy in these conditions, except those marked with an asterisk. The first three conditions demonstrate X-rav hypersensitivity, the remainder, UV hypersensitivity. Cellular hypersensftivity to UV is not seen in all cell-based studies in dysplastic naevus syndrome.
3. DNA repair and DNA repair genes After exposure to a wide variety of DNA-damaging agents, as part of a cellular survival response, many genes become transcriptionally active (ie. synthesise messenger RNA). The majority of these genes are not directly involved in repairing the damaged DNA, however in the last few years a number of human repair genes have been identified, most of which are involved the the repair of damage caused by UV radiation. Largely because of the complexity of X-ray induced damaged, there has to date been only one such X-ray repair gene identified, XRCC- 1 (X-ray Repair Cross Complementing Chinese hamster ovary cells). Although the function of XRCC-1 is largely unknown, it is known to be associated with the repair of single strand DNA breaks. New strategies to further study the biologic function of XRCC-1 include the construction of transgenic mice, deficient in the function of this gene (Larry Thompson; Lawrence Livermore National Laboratory, Livermore, USA). The biological relevance of the cloned repair genes is graphically illustrated by those human disorders resulting from the lack of function of a single one of these genes. For example, Christine Weber (Lawrence Livermore National Laboratory, Livermore, USA) reported that her group have recently demonstrated that a UV repair gene, ERCC-2 (Excision Repair Cross Complementing Chinese hamster ovary cells), fully corrects the repair defect in cell lines from one subset of xeroderma pigmentosum (XP) individuals [ie. ERCC-2 is the gene deficient in this condition (XP-D)]. Previously, groups from Japan and the Netherlands had shown that genetic complementation of other XP subsets was possible, using two of the other five cloned ERCC genes. All six ERCC genes have been isolated using rodent systems. Australasian Radiology. VoL 36. No. I , Fehruai?. 1992
9TH INTERNATIONAL CONGRESS OF RADIATION RESEARCH, TORONTO, CANADA, 7-12 JULY 1991
These studies thus underscore the contribution of such laboratory models to an understanding of human genetic diseases. Dirk Bootsma (Erasmus University, Rotterdam, The Netherlands), gave an overview of the present state of knowledge regarding the six ERCC genes. All six genes encode proteins involved in the incision step of UV excision (‘patch’) repair. Despite this, the genes vary significantly in size, and code for protein sequences which would, on the basis of structure alone, be predicted to have diverse biological roles. As was indicated earlier, Phillip Hanawalt (Stanford University, Stanford, USA) stressed that, at least as far as UV radiation was concerned, actively transcribing genes demonstrate preferential repair. This pattern has held true for every human locus examined to date. No such data is presently available for ionising radiation. More recently, it was shown that even within preferentially repaired genes, lesions on the DNA strand which was actively transcribing ( t o mRNA), were more efficiently removed. Futhermore, there is evidence that UV-induced damage to inactive genes in the immediate vicinity of a preferentially repaired, active gene, can enjoy augmentation of repair, implicating a regional repair effect. The basic experimental substrate for the isolation of human DNA repair genes have been rodent cell ‘mutants’, deficient in one or more aspects of DNA repair. In the case of the cloned repair genes, the rodent cell deficiency was due to absence or inactivation of that particular gene. The addition of human DNA complemented the rodent defect. This allowed the respective genes to be identified. Over 20 such X-ray repair-deficient rodent strains have been isolated and characterised by multiple laboratories, since 1983. Three more such variants were described, by Murray Stackhouse (Colorado State University, Fort Collins, USA), A. Varghese (Ontario Cancer Institute, Toronto, Canada) and by a Sydney University group (MM). As an alternative approach to the study of genetic factors governing radiosensitivity, Jack Russell (Glasgow, UK) reported the isolation of a radioresistant neuroblastoma cellular variant from the repeated exposure of a radiosensitive parent line to X-irradiation. It is possible that such a variant may demonstrate, for example, amplification of genes involved in DNA repair. Discussing the enzymology of DNA repair in E. coli, Susan Wallace (University of Vermont, Vermont, USA) (President Elect of the Radiation Research Society) emphasised that DNA base excision repair enzymes, responsible for repair of qualitatively minor X-ray induced lesions, were ubiquitous, highly conserved proteins. Identification and molecular cloning of such bacterial genes was thus relevant to the isolation of their human homologues (equivalents). Three such genes have been cloned to date.
4. Molecular genetics of tumour progression Webster Cavanee (Montreal University, Montreal, Canada) presented compelling molecular evidence for the biological progression of low grade, through intermediate grade, to high grade cerebral astrocytomas. For example, in over 120 glioblastomas multiforme examined, over 95% had a constant abnormality associated with chromosome 10 (a deletion), found in none of the lower grade tumours. All grades of astrocytoma examined were associated with another fixed abnormality of chromosome 17 (also a deletion). Epidermal growth factor receptor (EGFR) was amplified preferentially in intermediate, and Australasian Radiology Vol.36. No. I. Fehruap, 1992
to a lesser extent, high grade tumours. These facts alone should facilitate more accurate diagnosis of these tumours. Perhaps more importantly, they strongly suggest an ordered succession of genetic abnormalities during a progression from low to high grade cancers, confirming an observation first made in relation to colorectal cancer. As such, gliomas may also prove a useful model for tumour progession at other sites. A further point was that the degree of aggressiveness of a number of glioma cell lines (as measured by invasion into foetal mouse brain) was reduced by successful transfection (transfer) of the missing chromosomal material into the glioma line, indicating the possible therapeutic application of the identification of deleted/inactivated tumour supressor genes (TSG’s) in cancers of different sites.
5. X-ray induced gene expression There are described an emerging number of genes, which are inducible by X-rays and other DNA damaging agents, as discussed by A1 Fornace (National Cancer Institute, Maryland, USA). In relation to X-rays, one general group is activated via a protein kinase C (PKC) pathway. These genes include the well characterised ‘immediate early’ genes: eg. jun, fos, E G R l , tumour necrosis factor, and a group of genes involved in negative growth control (eg the gadd-Growth Arrest and DNA Damage inducible-genes). In general, the study and characterisation of these sorts of genes may lead to the identification of those which are associated with intrinsic cellular radioresistance, however, to date there have been major difficulties distinguishing the genes of interest from the high background of induced genes not directly participating in DNA repair.
6. Oncogene activation during X-ray induced cellular transformation It is well established, both in the laboratory and clinic, that X-rays can induce the development of malignant cellular characteristics in benign cells. Analagous to the situation demonstrated at a number of other sites [notably the large bowel and more recently, for cerebral gliomas (see section 4)] the mechanism of this effect presumably involves a mutistep activation of dominantly acting cellular protooncogenes, in association with the inactivation of growth inhibitory (‘tumour supressor’) genes. The exact sequence of mutational events, however, remains to be shown, and may be different for different tissues. Using mouse 3T3 cells, Jack Little (Harvard University, Boston, USA) and colleagues examined cell colonies transformed after X-rays (6Gy), for evidence of activation of oncogenes, using Southern analysis, as well as a more sensitive technique (denaturing gel gradient electrophoresis). All 15 dominantly-acting oncogenes examined, including the Ras family of proteins, showed no induction when compared with controls. The p53 protein, which has both dominant and recessive (tumour supressive) effects, was mutated in approximately 20% of the clones examined. Further attempts to elucidate molecular concomitants of neoplastic transformation by X-rays are proceeding. In summing up, Professor Little hypothesised that because genetic recombination (a normal event at meiosis) was a major repair pathway for DNA double strand breaks (critical X-ray induced lesions), activation of a general recombination machinery by X-rays may account for their substantiaf transforming ability, by altering the normal spatial relationship between oncogenes and their promoters/ enhancers. This could result in altered gene expression. The actual oncogenes implicated require definition. 87
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7. Cell cycle biology and radiation There has been considerable recent interest in cellular proteins which induce, or are associated with, the various cell cycle phases. Whilst it has been recognised for some time that the G2-M boundary was an important checkpoint, in that, once passed, the cell was committed to mitosis, its molecular basis has until recently, been unclear. Joseph Roti-Roti (St Louis, USA) reported on studies in yeast which have recently identified a number of specific protein kinases which appear to mediate this effect. Radiation sensitive yeast mutants have shown that the lack of a radiation-induced G2-M block greatly reduces the fidelity and completeness of DNA repair after X-irradiation. These yeast proteins show a strong homology to human proteins with the same function. One of the basic cellular defects in cells from individuals with ataxia telangiectasia is an enhanced cellular accumulation at the G2-M boundary after X-irradiation. Whether these proteins or their regulation is abnormal in this condition is presently being evaluated. Reporting on studies evaluating G2 phase delay after ionising radiation, W. Gillies McKenna (University of Pennsylvania, Pennsylvania, USA) pointed out that the appearance of the mRNA for a specific protein kinase, cyclin B, was associated with the end of the normal G2 delay in mammalian cells. He postulated an addition to the list of possible contributors to intrinsic cellular radioresistance, namely, inhibitors of, or defects in, cyclin €3 expression, which may thereby allow greater damage processing before a cell enters mitosis.
8. Proton beams in radiation therapy I n the Ontario Cancer Treatment and Research Foundation (Richards) Award lecture, Herman Suit (Massachusetts, USA) provided an overview of the experience of the Massachusetts General Hospital and its collaborators with proton beam therapy since 1972. Although the clinical spectrum of diseases for which this modality is useful or appropriate is limited, impressive results have accrued in the treatment of uveal melanomas and sarcomas of the skull base. In the case of the former, over 1600 patients have been treated, with 5 year local control within the high dose volume of 99% (and an overall local control of 96%), and 5 year survival of approximately 80%. Preservation of vision in the vast majority of patients is the obvious advantage over surgery. These figures are mirrored in the other two functional proton facilities (one US, one French, having treated approximately 350 and 900 patients respectively). 9. Assays predictive of radiation response There is great interest in developing clinically relevant assays of tumour (and normal tissue) radiosensitivity. Such predictive assays may help define which subsets of tumours may be better treated with altered therapies such as concurrent chemotherapy or accelerated fractionation. For example, tumours with short doubling times may be better treated with the latter approach.
a. In vitro clonogenic cell survial assays The surviving fraction of cells after 2Gy (SF2) has been shown to correlate in some cases with variations in clinically observed tumour radiosensitivities. This test involves developing a primary tumour cell culture from a biopsy and as such is relatively slow, laborious and costly. On average, 70% of biopsies taken develop into usable cultures. Early data are available on two assays, tha CAM clonogenic assay and the Courtney clonogenic assay.
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William Brock (MD Anderson Cancer Center, Houston, USA), whose group developed the CAM assay, presented 140 patients with head and neck (H & N) cancer who received post-operative radiation therapy. SF2 ranged from 0.10 to 0.92, with a mean of 0.32. They found no significant correlation between SF2 and local control but their results are difficult to interpret because surgery was the primary treatment modality, T. Girinsky (Institute Gustave-Roussy, Villejuif, France) showed information on 97 H & N and cervix tumours treated primarily by radiation therapy, using the CAM assay to calculate SF2. A cell growth fraction was also calculated by flow cytometry using cells labelled with bromodeoxyuridine. The mean SF2 for cervix and H & N cancers was similar (0.39 and 0.38 respectively). Tumours with SF2
