STUK-A100 APRIL 1991
Biological effects of radiation: — — The induction of malignant transformation and programmed cell death
Kristina Servomaa STUK-A100 APRIL 1991
Biological effects of radiation: The induction of malignant transformation and programmed cell death
Kristina Servomaa Finnish Centre for Radiation and Nuclear Safety Department of Genetics, University of Helsinki
Academic dissertation
To be presented, with the permission of the Faculty of Science of the University of Helsinki, for public criticism in Auditorium XII on April 26th, 1991, at 12 o'clock noon.
FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY P.O.Box 268 SF-00101 HELSINKI Finland Tel.+358 0 70821 ISBN 951-47^*533-1 ISSN 0781-1705
The Finnish Government Printing Centre P.O.Box 516 SF-O01O1 HELSINKI Helsinki 1991
Sold by: The Finnish Government Printing Centre P.O.Box 516 SF-00101 HELSINKI Tel. +358 056601 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
SERVOMAA, Kristina. Biological effects of radiation: The induction of malignant transformation and programmed cell death. SJUK-A100, Helsinki 1991,55 p. + app. 67 p.
ISBN 951-47-4553-1 ISSN 0781-1705 Key words: radiation, carcinogenesis, oncogenes, apoptosis, LINE, hot particles, Chernobyl SUMMARY
Modem molecular biology has led to new insights for a better understanding of the mechanisms underlying radiation-induced carcinogenesis and cell death. One of the most interesting questions concerns the role of oncogenes, anti-oncogenes, and mobile genetic elements in malignant transformation. UV and ionizing radiation are DNA-damaging agents. The mutagenic, cytogenetic, carcinogenic,. nd lethal events in cells exposed to radiation are the result of primary damage to DNA. The selection and multiplication of nonlethally repaired DNA damage via cell division is a particularly important aspect of carcinogenesis. The potentially most hazardous factors that enhance this process are those that result in the sustained proliferation of irradiated cells (promotion by mitogenesis). Another mechanism involves a remodelling of the genome by insertional mutagenesis.
!n the Chernobyl explosions and fire, powdcrized nuclear fuel was released from the reactor core, causing an unexpected fallout. X-ray analysis and scanning electron microscopy showed that the isolated single particles were essentially pure uranium. These uranium aerosols contained all of the nonvolatile fission products, including the B-emitters, wZr, 103Ru, ,06Ru, I41Ce, and I44Ce. The hot panicles are extremely effective in inducing malignant transformation in mouse fibroblast cells in vitro. The major factor responsible for this effect is focus promotion caused by a wound-mediated permanent increase in cell proliferation (mitogcnesis associated with mutagenesis).
Transformed foci were analysed for the activation of c-aol, c-erb-A, c-erb-B, c-/ms, c-fos, c-myb, c-myc, c-lla-ras, c-Ki-ras, c-sis, and c-ra/oncogenes at the transcriptional level. The pattern of oncogene activation was found to vary from focus to focu.i.
Long interspersed repeated DNA (LI or LINE) makes up a class of mobile genetic elements which can amplify in the cell genome by retroposition. This element is spontaneously transcriptionally activated at a critical population density and later amplified in rat chlorolcukacmia cells. UV light and ionizing radiation induce this activation prematurely, and the activation is followed by programmed cell death (apoptosis) in a sequence of events identical to that seen in LIRn activation occurring spontaneously.
The radiation-induced activation of the L/ element is based on, or at least associated with, direct DNA damage. The observed large-scale activation of LI elements creates a burst of lethal mutations. Radiation might also lead to the small-scale rctroposition of the LI element in the genome and, hence, result in potentially hazardous sublethal mutations in celts. If radiation induced primary DNA damage could sometimes lead to complex mechanisms via rctroposiiions of LI, it would alter the current opinions of radiation-induced health hazards.
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SERVOMAA, Kristina. Säteilyn biologiset vaikutukset: Pahanlaatuinen transformaatio ja ohjelmoitu solukuolema. STUK-A100, Helsinki 1991,55p.+ luit. 67 s. ISBN 951-47-4553-1 ISSN 0781-1705
Avainsanat: säteily, syövän syntyminen, syöpägeenit, apoptoosi, LINE, kuumat hiukkaset, TSemobyl YHTEENVETO Molekyylibiologian (geeniteknologian) menetelmien nopea kehittyminen on tehnyt mahdolliseksi säteilyn aiheuttaman syövän syntymiseen ja solukuolemaan liittyvien mekanismien tarkemman tutkimisen. Tärkeänä kohteena ovat syöpägeenit, tuumorciden syntymistä estävät geenit (ns. aniionkogeentt) ja liikkuvat geneettiset elementit. UV- ja ionisoiva säteily voivat aiheuttaa vaurioita DNA-molekyylissä ja tällainen DNA:n primaarinen vaurio aiheuttaa mutaatioita, sytogeneettisiä muutoksia, syöpää ja solukuoleman. Syövän kehittymisen kannalta on erittäin merkityksellistä, että DNA-muutos valikoituu ja monistuu solun jakautuessa, joten solujen jatkuvan jakautumisen aiheuttavat tekijät ovat potentiaalisesti vaarallisimpia vaurion ilmitulon kannalta. Epäsuora mekanismi, joka voi johtaa syöpään ja solukuolemiin, on genomin uudelleenjärjestäytyminen.
Täemobylin ydinvoimalaturmassa kova paineja räjähdykset murensivat osan ydinpolttoaineesta pieniksi kappaleiksi. Näin syntyneet ns. kuumat hiukkaset aiheuttivat odottamattoman päästön levitessään ilmavirtojen mukana luontoon. Alkuaineanalyysi osoitti näiden hiukkasten olevan puhdasta uraania. Gammaspektrometrisissä mittauksissa todettiin, että nämä hiukkaset sisälsivät kaikki haihtumattomat fissiotuotteet, esim. B-säteilijät 95Zr, ,03Ru, ,wRu, ,4ICe ja 144Ce. Kuumat hiukkaset aiheuttivat erittäin tehokkaasti pahanlaatuisia transformaatioita hiiren sidekudossoluviljelmissä (C3H 10T1I2). Kuuma hiukkanen aiheutti ympäristössään solukuolemaa niin runsaasti, että siihen syntyi pysyvä haava, jonka ympäristön solut yrittivät parantaa jakautumalla jatkuvasti aiheuttaen DN A-vaurion promootion (mutageneesia seuraava mitogeneesi). Transformoituneista solupesäkkeistä tutkittiin onkogeenien c-abl, c-erb-A, c~erb-B, c-fms, c-fos, c-myb, c-myc, c-Ha-ras, c-Ki-ras, c-sis ja c-raf aktivaatiota. Minkään tietyn onkogeenin tai onkogeeniryhmän aktivaatio ei ollut säännönmukaista, vaan pesäkkeet erosivat toisistaan.
Nisäkkäiden genomissa on hajallaan toistuvia geneettisiä DNA-jaksoja ns. LINE- eli /-/-elementtejä, jotka voivat monisrua retroposition avulla. Tämän elementin transkription on todettu aktivoituvan spontaanisti rotan kloroleukemiasoluissa tietyssä solutiheydessä solujen kasvaessa eksponentiaalisesti. Myöhemmin /-/-elementti monistuu soluissa. UV-ja ionisoiva säteily aiheuttavat varhennetun aktivaation ja tätä aktivaatiota seuraa ohjelmoitu solukuolema samaan lapaan kuin spontaanin aktivaation jälkeen.
Säteilyn aiheuttama /-/-elementin aktivaatio perustuu tai ainakin se liittyy DNA-molekyylin vaurioitumiseen. Havaittu /-/-elementin aktivaatio aiheuttaa suuren määrän letaalimutaatioita, mutta säteilyn aiheuttama retroposilio voi olla määrällisesti huomattavasti pienempi tapahtuma, jolloin /,7-retropositio genomiin aiheuttaa potentiaalisesti haitallisia subletaaleja muutoksia soluissa. Jos tällainen säteilyn aktivoiman /-/-retroposition aiheuttama primaarinen DN A-vaurio olisi yleinen, se voisi muuttaa yleisiä käsityksiä säteilyn aiheuttamista terveysvaikutuksista.
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CONTENTS page
SUMMARY 3 YHTEENVETO 4 ORIGINAL PUBLICATIONS 7 LIST OF ABBREVIATIONS 8
1 INTRODUCTION 9
2 REVIEW OF THE LITERATURE 11
2.1 Biological effects of radiation 11 2.1.1 Primary damage 11 2.1.2 Chromosome aberrations 13 2.1.3 Malignant transformation 14 2.1.4 Cell death 16 2.2 Oncogenes 17 2.2.1 Groups of oncogenes 17 2.2.2 Activation of oncogenes 18 2.2.3 Expression of oncogenes in radiation-induced tumours 19 2.3 Mobile genetic elements 20 2.3.1 Structure and function of LINE (LI) 20 2.3.2 Reverse transcriptase 21 2.3.3 Rat LINE (LIRn) 22 2.4 Programmed cell death (apoptosis) 22 2.5. Hot particles 24
3 AIMS OF THE STUDY 25
4 MATERIAL AND METHODS 26
4.1 Cell culture 26 4.2 Cell morphology 26 4.3 Irradiation 26 4.3.1 X-irradiation 26 4.3.2 Irradiation by ^Co gamma rays 26 4.3.3 UV irradiation of chloroma cells 27 4.3.4 Hot particles 27 4.4 Gammaspcctrometric analyses 27 4.5 Plasmids and probes 27 4.6 Nucleic acid analyses 28 4.7 DNA fragmentation studies 29 4.8 In vitro transformation assays 30 4.9 Scanning electron microscopy 30 4.10 Time-lapse vidcography 30
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page
5 RESULTS AND DISCUSSION 31
5.1 Hot particle characteristics 31 5.2 Biological effects of radiation 32 5.2.1 Malignant transformation 32 5.2.2 Radiation-induced wounds and the role ofmitogenesis 3* 5.2.3 Oncogenes 35 5.3 Programmed cell death 38 5.3.1 Growth and morphology of chloroma cells 38 5.3.2 DNA fragmentation 39 5.3.3 Transcriptional activation of LIRn 40 6 CONCLUDING REMARKS 41
ACKNOWLEDGEMENTS 42 REFERENCES 43
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ORIGINAL PUBLICATIONS
The present thesis is based on the following original articles which are referred to by then- Roman numerals, I-VII, in the text. Some previously unpublished data is also presented in the text.
I RYTÖMAA T., SERVOMAA K., TOIVONEN, H. Chernobyl reactor accident and radioactive particles found in Finland. Duodecim 1986; 102:1190-1196 (in Finnish).
II TOIVONEN H., SERVOMAA K.t RYTÖMAA T. Aerosols from Chernobyl: particle characteristics and health implications. In: von Philipsbom H. and Steinhäusler F., eds. Hot Particles from the Chernobyl Fallout. Theyem: Schriftenreihe des Bcrgbau- und Industriemuseum, 1988; 16:97-105.
III SERVOMAA K., RYTÖMAA T. Activation of oncogenes by uranium aerosols: an in vitro study. In: Brustad T., Langmark F., Reitan J.B., eds. Radiation and Cancer Risk. New York: Hemisphere (Cancer Series), 1989:31-42.
IV SERVOMAA K., RYTÖMAA T. Malignant transformation of mouse fibroblasts by uranium aerosols released from Chernobyl. In: Riklis E., ed. Frontiers in Radiation Biology. Weinheim: VCH, 1990:589-594,
V RYTÖMAA T„ SERVOMAA K. Identification of a putative growth inhibitor gene in rat chloroleukaemia cells. In: Baserga R., Foa P., Metcalf D., Pölli E.E., eds. Biological Regulation of Cell Proliferation. New York: Raven Press, 1986: 93-101.
VI SERVOMAA K„ RYTÖMAA T. Suicidal death of rat chloroleukaemia cells by activation of the long interspersed repetitive DNA element (LIRri). Cell Tissue Kinct. 1988; 21: 33-43.
VII SERVOMAA K., RYTÖMAA T. UV light and ionizing radiations cause programmed death of rat chloroleukaemia cells by inducing retropositions of a mobile DNA element (LIRn). Int. J. Rad. Biol. 1990; 57; 331-343.
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LIST OF ABBREVIATIONS
A adenosine ATCC American Type Culture Collection BME Basal Medium-Eagle's bp basepatr(s) BSA bovine serum albumin Bq becquerel C cyudine cDNA complementary deoxyribonucleic acid cpm counts per minute dCTP deoxycyudine triphosphate DMEM Dulbecco's modified Eagle's medium DNA deoxyribonucleic acid dsb double-strand break DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor EMBL European Molecular Biology Laboratory FCS fetal calf serum Gy gray IAP intracisternal A-particle kb kilobase(s)orlOOObp kbp kilobasepair LI, LI fin LINE (Rn = Rattus norvegicus) LINE long interspersed nuclear element LET linear energy transfer LTR long terminal repeat MuSV murine sarcoma virus OD optical density ORF open reading frame PBS phosphate buffered saline PDGF platelet-derived growth factor RNA ribonucleic acid RNaseA ribonuclease A SDR short direct repeat SDS sodium dodecyl sulfate SINE short interspersed nuclear element ssb single-strand break SSC 0.15 M NaCl/0.015 M Naa-cilrate, pH 7.6 SEM scanning electron microscopy T thymidine TE 10 mM Tris-HCl (pH 8), 1 mM EDTA UV ultraviolet
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1 INTRODUCTION
The cytotoxic, carcinogenic, and mutagenic events in cells exposed to radiation are thought primarily to be the result of damage to DNA. Damage to the genome, resulting in critically altered genetic information, may be induced directly or indirectly. It is essential to realize, however, that the harmful biological effects of ionizing and UV radiations cannot be understood or described solely on the basis of interactions of radiation with matter.
Both DNA damage (mutagenesis) and mitogenesis are important aspects of carcinogenesis. Damage to the single genome is amplified only when the damage is transmitted from cell to cell. The most obvious amplification mechanism is, of course, multiplication through cell division, but other hypothetical mechanisms, such as through the activation of a provirus, are also possible, even if unlikely. Expression of the damage through the selection and amplification mechanisms may be promoted or suppressed by environmental factors unrelated to radiation. With regard to these environmental factors, experimental evidence shows that simple manipulations, such as forcing irradiated cells into maintained proliferation, can dramatically increase the expression of a transformed phenotype.
The most remarkable biological effects of radiation on cells are die induction of mutations, the conversion to a (pre)cancerous state, and death. It is apparent that a small dose is able to induce a cell to become malignant, but before this can result in a clinical tumour, the cell and its progeny must be capable of continuous division over many generations. On the other hand, small doses are much less effective than large ones in killing cells. It seems that an accumulation of misrepaired damage or a failure to repair critical lesions is required before cell death is induced. Considerable evidence points to nucleotide base damage and to single- and double-strand breaks in DNA molecules as the most critical lesions for cell death. In particular, nonrepaired and faultily repaired double-strand breaks lead to major asymmetrical chromosomal aberrations that cause the mitotic death of cells. Programmed cell death (apoptosis) is commonly observed under physiological conditions when cell death is cither beneficial or a controlled response to mildly pathological or sublethal stimuli which by themselves would not induce direct cell death. There is reason to believe that radiation may activate the programme leading to suicidal cell death.
Among the rctroclcments which may have the capacity to transpose and induce cell damage are nonviral elements, such as UNEs {LI, long interspersed nuclear element), and retroviruslikc elements with structural analogies to infectious retroviruses. Indeed, the contemporary transposition of LINEs has been shown to take place in numans, causing disease by insertional mutation. LINE represents a specific class of nonviral retrotransposons that can be dispersed in the genome, evidently through an RNA intermediate, causing disease or cell death by insertional mutations). The transcriptional activation of rctroposablc cellular elements can lead to various insertional mutations that differ in their frequency of occurrence and location within the genome. The outcome of this
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type of genomic remodellingevidentl y depends on clonal selection and amplification of the cell, and it can also be lethal to the cell. From the point of view of clonal selection and amplification, the potentially most hazardous factors are those that result in die sustained proliferation of irradiated, critically altered cells.
Therefore, the series of experiments described in this study focus on the radiation-induced activation of cellular programmes leading to malignant transformation or cell death. It is commonly thought that radiation-associated carcinogenesis and cell death are simply based on primary DNA damage, where only the extent and location of die damage vary. However, die two processes may also evolve another way: both may be induced by activated mobile genetic elements. This viewpoint underlies die motivation for the present study, although die actual experiments also address more apparent and practical issues, such as the effects of Chernobyl-associated nuclear fuel particles on cells.
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2 REVIEW OF THE LITERATURE
2.1 Biological effects of radiation
A variety of lesions can be detected in the DNA of eukaryotic cells irradiated with ionizing and ultraviolet radiation. Exposure of cells to these forms of radiation can result in genetic mutations, chromosomal aberrations, oncogenic transformations, and cell death. The types of DNA damage in the irradiated cell and the accompanying mechanisms of DNA repair are essential topics in radiation biology. Primary DNA damage includes single- and double-strand breaks of DNA, DNA-protein crosslinks, base alterations and base detachments, sugar alterations, and bulky lesions (i.e., clusters of base damage).
The oncogenic transformation of cells is a multistep process in thata series of specific events is required to transform a normal cell into a malignant one (Barrett and Fletcher 1987, Borek 1988). The multistep model of carcinogenesis involves the initiation of irreversible genetic alterations and the promotion of clonal populations of initiated cells which expand and ultimately progress to malignancy (Borek 1985). The initiating events are not well understood. Initiation is thought to involve some type of heritable change which predisposes the affected cell and its progeny to subsequent neoplastic transformation (Borek 1987a, Borek 1988, Farber 1984). It has been postulated that the transformation of cells may involve the activation or abnormal expression of oncogenes (Guerrero et al. 1984, Guerrero anc Pellicer 1987) and that more than one of these may be activated to induce cancer in vivo (Balmain 1985, Land etal. 1983a). On the other hand, in some cancers, the inactivation of anti'Oncogenes is involved (Stanbridge and Nowel! 1990, Weinberg 1988). Tumour promotion is the process through which the initiated cell and its progeny subsequently undergo additional change(s) necessary for neoplastic transformation. The final expression of cancer in vivo may be determined by several interacting factors (Fry 1981, Upton 1984) and may involve more than one step, typically requiring repeated and prolonged exposure to the promoting stimuli (Upton 1984).
Accordingly, the exact nature of radiation-induced malignant transformation is not clear at present, and it may also vary from case to case. While DNA is the ultimate target in radiation carcinogenesis, the frequency and subsequent course of neoplastic processes are determined by an interplay of radiation-dependent and -independent, endogenous and exogenous factors (for a review, see Borek 1987b).
2.1.1 Primary damage
As already noted, the most important target in the nucleus is DNA. Damaged structures in DNA can be classified as singly modified bases, single-strand breaks, double-strand breaks, large structural modifications, and DNA-protein adducts. The induction of point mutations
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- that is, changes in die molecular structure (base sequence) of the DNA-appears to occur at a rate that is directly proportional to the dose (Furuno-Fukushi and Matsudaira 1989).
Damage to the genome, resulting in critically altered genetic information, may be induced directly or indirectly. Ionizing radiation has been postulated to induce multiple biological effects by its direct interaction with DNA or through the formation of free radical species which then lead to DNA damage (Hall 1988). It is essential to realize, however, that die harmful biological effects of ionizing radiation cannot be understood or described solely on this basis.
Ionizing radiation causes the frequent formation of strand breaks in cellular DNA. Particularly, „ie double-strand break has been identified as the lesion most likely to be the cause of die lethal effects of ionizing radiation (Painter 1979, Ward 1988). The number of DNA double-strand breaks is linearly related to the radiation dose in the presence of oxygen (low-LET radiation). In addition, the yields of DNA damage per dose among different cell types are constant (Ward 1990). After cobalt gamma ray and X-ray irradiations, die ratio of DNA double-strand breaks to single-strand breaks is about 1 to 10 (Roots et al. 1985). If this estimate is correct, a substantial number (about 100 or more) of DNA double-strand breaks are formed in a mammalian cell after moderate doses (1 Gy) of radiation (Baverstock and Will 1989, Goodhead 1989). Although it is informative to know about the initial number of DNA strand breaks, it is of greater importance to have an understanding of die numbers of nonrepaired or faultily repaired DNA breaks of various kinds. Base substitutions represent about 2/3 of the point mutations in mammalian cells.
It should be noted that die linear relationship between dsb's and dose suggests direcdy diat even this damage is caused by a single photon and not by a double hit. Furthermore, it can be easily calculated from the frequency of dsb's per photon fluence that the chance of two photons hitting die same DNA target, wiUiin a reasonably short length of die molecule, is negligible at die dose levels relevant to human exposure (Servomaa and Rytömaa 1990; see also ICRP 581990, Goodhead 1989).
UV radiation (along with several other forms of radiation) is known to be capable of causing direct DNA damage. Photons of UV-C (<290 nm) and UV-A (>320 nm) have very different biological effects and mechanisms of activation; UV-B (290-320 nm) effects are a mixture of die UV-A and UV-C types. The most common type of damage by UV is probably die production of dimers between adjacent pyrimidine residues and (6- 4)photoproducts which can interfere with both DNA replication and transcription (Trgovcevic* et al. 1989). Pyrimidine dimers occur dirough die symmetric reaction of die 5,6 bonds of any two adjacent pyrim idines in a DNA strand. The (6-4)photoproducts are formed by a stable bond between positions 6 and 4 of a "TpC", but not a "CpT", doublet (Franklin etal. 1982). UV-A imposes DNA damage less efficiently dian equivalent fluencerate s of UV-B. The yields of pyrimidine photoproducts due to UV-A are smaller by orders of magnitude dian die yields obtained from die shorter wavefengdi UV-B. Oiher types of DNA damage more characteristic to UV-A are DNA strand breaks, alkali-labile sites, and DNA-to-protein
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crosslinks. UV-A is capable of both making and breaking covalent bonds. Radiation of wavelengths as long as S20 ntn can induce single-strand breaks (Peak and Peak 1989).
2.1.2 Chromosome aberrations
When cells are exposed to ionizing radiation, one of the most readily noted effects is the production of aberrant chromosomes, particularly observable when the cells are in the metaphase of mitosis (IAEA 1986). These aberrations are the result of either simple breakage of the chromosomes or the interaction of the broken ends to form rearrangements. They can be classified as two different types, according to die stage of the cell cycle during which exposure takes place. If the cells are exposed during G i, before die DN A is replicated, the unit ot DiCikage and rejoining is the whole unreplicated chromosome. If S or G2 phase cells are irradiated, the individual chromatid is die unit of breakage and rejoining, which leads to the formation of chromatid aberrations. Most of the breaks produced by radiation are repaired. A small proportion, however, is either faultily repaired or not repaired, leading to the formation of fragmented chromosomes or rearrangements that can be seen and analysed.
To understand radiation's effects on cells, it is paramount to realize diat repairing die primary damage does not necessarily mean that die original structure (base sequence) will be faithfully reproduced. For instance, with respect to me dsb, repair simply means rejoining a broken end with another broken end (see Comfonh et al. 1989), but no evidence has ever been published which would suggest diat an alteration in base sequence is unlikely to occur during the rejoining. Intead, it has been speculated that mobile DNA elements, such as LINE (see later), may participate in die repair of dsb's (Edgell et al, 1987. Voliva et al. 1984), and mat mis would naturally alter die base sequence, even in cases where rejoining does not involve 'foreign' DNA ends. Regarding certain chromosome aberrations commonly induced by radiation, such as die formation of dicentric chromosomes, it is also important to realize that a broken DNA end is not rejoined with an intact chromosome (intact telomere; see Cornforth et al. 1989).
The total yield of chromosomal aberrations induced by comparable radiation doses in mammalian lymphocytes, including in vivo exposure in humans, is an order of magnitude higher man the observed frequency of malignant transformation in vitro (e.g., Lloyd and Edwards 1983). The conventional measurement of a mutation leads to a serious underestimation of the actual mutational efficiency of radiation because cell death eliminates cells that have developed or accumulated lethal mutation?! insults from die scoring process (Waldren et at. 1986).
An unanswered question is whether thechromosome?Jterations observed in malignant cells are die cause of die malignancy or a by-product of die agent initiating die cancer. It has been hypothesized that gene rearrangements, resulting ultimately from double-strand breaks, may be critical events in radiation-induced cancer (Chadwick and Leenhouts 1981). Chromosomal alterations found in tumour cells can include numerical changes or structural
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changes, or both. Chromosomal translocations of the type that are known to be produced by radiation have been shown to result in the activation of the myc oncogene by promoter insertion in Burkitt's lymphoma (Aisenberg 1984). It may also be worth noting here that the Philadelphia chromosome (a translocation between chromosome 9 and 22; t(9;22Xq34;ql 1)), involving the activation of the c-ubl oncogene (de Klein et al. 1982), is probably directly associated with the development of chronic myeloid leukaemia (Kamada and Uchino 1978), and that this disease is a particularly common malignancy induced by radiation in man (Preston et al. 1987, Kato et al. 1989).
Rearrangements and deletions may be the initiating events for myeloid leukaemia induced by radiation in mice. These chromosomal abnormalities are a common feature of all, or almost all, of the myeloid leukaemias examined and were observed as an early event. Many radiation-induced osteosarcomas, perhaps up to 30 per cent, display an amplification of a defined region of chromosome 1S depending on the mouse strain (Janowski et al. 1990).
2.1 J Malignant transformation
Tumours are not observed in vivo until an advanced stage is reached. The early events that take place after irradiation and/or during irradiation are of particular importance and may be determined in an in vitro system. These events include the activation of oncogenes and proviral genes, and their impact on cell proliferation and differentiation. Malignant transformation in vitro by radiation is also a multistep process initiated by the induction of a mutation. The production of the damage is followed by repair, and then the fixation of the mutation and expression of various transformed phenotypes during cell replication and the subsequent confluent state (Balcer-Kubiczek et. al. 1987; Borek and Sachs 1968; Kakunaga 1974, Suzuki et al. 1989, Terasima et al. 1981),
The control of cell proliferation is lost in cancer cells which proliferate in vivo at inappropriate times and locations in the body. Cells progress to stages of greater malignancy characterised by more rapid net growth, immortalization, invasiveness, metastasis, and induced angiogenesis. The escape of cancer cells in vitro from growth control is evidenced by their ability to grow in culture in low concentrations of serum (i.e., in the absence of exogenous growth factors) and without anchorage dependence. The transformed cells can either produce their own growth factors, for which they also express the corresponding receptors, or they may bypass the control of proliferation through deregulation of signal transduction pathways (Druker et al. 1989, Weber and McOure 1987).
The ultimate aim of all in vitro transformation studies is to elucidate in vivo responses and mechanisms using simple systems which can be studied and manipulated easily. The transformation of mammalian cells in vitro provides quantitative and qualitative information on the processes by which physical and chemical agents induce malignancy. Cell cultures provide powerful models for investipting the process of radiation-induced malignant transformation under conditions free from host-mediated effects. Oncogenic transformation in in vitro cell cultures may represent an artificial system, however.
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Table 1. Commonly used criteria for transformation in vitro, i.e., criteria for a transformed phenotype (Wright 1989, Chan and Little 1986).
1. Altered cell morphology 2. Enhanced net growth rate 3. Increased saturation density because of loss of contact inhibition 4. Loss of anchorage dependence 5. Reduced serum dependence 6. Extended or infinite life span 7. Chromosomal abnormalities (diploid -» aneuploid) 8. Changes in cell surface antigens 9. Proteinase production 10. Loss of normal differentiation 11. Tumour production in a compatible host
Table 1 shows a list of properties of cells commonly regarded as transformed in vitro. One of the most important in vitro properties of the transformed cells is the loss of anchorage-dependent growth; i.e., the cells grow in a suspension culture without requiring attachment to the extracellular matrix. Furthermore, the transformed cells continue to grow to higher cell densities than nontransformed cells, which reveals the lack of contact inhibition and, possibly, reduced requirement for exogenous growth factors. In addition, fully transformed cells produce tumours in animals, such as nude mice.
The C3H10T1I2 transformation assay has changed little since its introduction seventeen years ago by Reznikoff et al. (1973a, b). It is based on the findings that transformed C3H 10T1I2 cells lose their strict contact inhibition and pile up to form distinct, densely staining, multilayered foci which are tumourigenic in animals and show anchorage independence. Three types of foci occur - types I, II, and III - which are identified by focus architecture and by the oncogenic potential of the cells, with type III being die most malignant (Reznikoff et al. 1973a). It is likely that the loss of contact inhibition following transformation is the result of changes in the plasma membrane structure. The C3H10T1/2 cells bear little resemblance to normal diploid fibroblasts, being aneuploid and immortal. In the current absence of a reliable human model, the C3H10T1/2 system remains one of the best characterised and most reproducible transformation assays available.
In terms of the initiation of the transformed phenotype by radiation, there is very good evidence that this event may be induced at a surprisingly high frequency in irradiated cultures of rodent cell lines (Kennedy 1985). The transformation curve shows a rise in frequency up to about 6 Gv. The graph then reaches a plateau (Little 1977). Low-dose work has shown that transformations could be detected even at very low doses, provided enough plates were set up (about 1000 per point for doses below 0.3 Gy; Miller and Hall 1978). Dose-response relationships were also obtained for a-particles (Lloyd et al. 1979), neutrons
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(Hill et ai. 1985), and for split-dose treatment (Terzaghi and Little 1976, Miller and Hall 1978). It has been shown that subculture after irradiation altered the number of foci obtained; in particular, subculture is extremely effective in enhancing the expression of a radiation-induced change if the number of foci is expressed per irradiated cell (Kennedy 1984, Kennedy and Little 1984).
The induction of these cellular effects has been found to be dose-rate-dependent; at low doses and low dose fates, the carcinogenic effectiveness of X-rays and gamma rays per unit dose is sometimes reduced, whereas that of high-LET radiation tends to remain constant or may even increase (Upton 1987). Also, the dose-rate dependence of radiation-induced mutations has been found to vary with the type of mutation and the nature of the DNA lesion induced (Upton 1987). On the other hand, however, some of the commonly accepted interpretations may be misleading. In particular, it is possible that in the conventional experimental setup in vitro, high doses and dose rates induce so much cell death that the surviving (and mutated) cells undergo an excess number of divisions and, therefore, "fix" die mutation more effectively or quicsiy than after low-dose/dose-rate irradiation. It has also been shown that the number of mutations produced by radiation is greatly underestimated in a conventional experimental setup because the indicator mutations are frequently associated with other lethal changes and, therefore, cannot be scored (Waldren et al. 1986). In in vivo conditions, radiation-independent promoters may actually determine the probability at which a malignant genotype is expressed.
These interpretations are supported by the finding that die density of cells plated post-irradiation has a marked effect on the observed transformation frequency; the frequency was reduced as the cell density increased (Han and Elkind 1979, Little 1979). This observation has been attributed to a requirement for a minimum number of cell divisions to occur in these irradiated populations before confluence is reached and the cells stop dividing. Such an explanation is reasonable for a cell system which exhibits true contact inhibition of cell division. Other explanations for this cell density effect may have to be explored, however, since the presence of certain "cellular factors" in the medium of high-density cultures could act as suppressors of transformation (Hall 1988).
On the basis of Uiese types of observations, Kennedy and Little (1980,1984; Kennedy 1984) proposed a two-step model in which die initial, radiation-induced event is frequent, occurring in a large proportion of cells. The second step is a rare event occurring at confluence or during the growth phase and it has a constant but small probability of occurring each time an initiated cell divides. It was suggested that this later event leads directly to the transformed phenotype.
2.1.4 Cell death
The strict definition of cell death means the loss of all functions and viability. After modest doses of radiation, however, mammalian cells may lose the capacity to proliferate but remain viable with respect to metabolic functions. Considerable evidence points to
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nucleotide base damage and to single- and double-strand breaks in DNA molecules as the most critical lesions in cell death (Radford et al. 1988). After irradiation, some cells die during the interphase (for apoptosis, see page 22), but mitotic death may be more common. In particular, nonrepaired and faultily repaired double-strand breaks lead to major asymmetrical chromosomal aberrations that cause the mitotic death of cells (Bryant 1984, Joshietal. 1982).
The amount of cell death is dependent on the type of radiation, the dose rate, the repair interval before division, and even on culture conditions. In the particular case of radiation transformation, the problems are accentuated by the relative weakness of radiation as a carcinogenic agent, probably due to the high level of cell death. A dose of 100 Gy is necessary to destroy cell functions in nonproliferating systems, such as nerve, muscle, or secretory cells (Hall 1988). For these differentiated cells, death can be defined as the loss of a specific function. By contrast, the mean lethal dose for the loss of proliferative capacity is usually less than 2 Gy (Hall 1988). For proliferating cells, such as haematopoietic stem cells and cells growing in culture, loss of the capacity for sustained proliferation is an appropriate definition of death (Hall 1988).
2.2 Oncogenes
An understanding of the molecular events leading to the development of cancer has progressed rapidly in the last few years. A major event has been the identification of cellular genes capable of inducing phenotypic changes characteristic of tumour cells. The genome in normal cells contains genes that exhibit transforming activity when properly activated. The viral oncogenes (y-cnc) are altered or overexpressed versions of their normal cellular proto-oncogene counterparts (c-onc). Around 60 such genes (proto-oncogenes) have now been identified, either as cellular homologies (c-onc) of the transforming genes of retroviruses (v-onc) or by their biological properties in transfection assays (Land et al. 1983a, Cooper and Lane 1984, Marchal and Rigby 1984,VarmusH. 1989, Weinberg 1985). In general terms, oncogenes are genes that are capable of inducing or maintaining cell transformation. They were originally discovered through the study of transforming retroviruses (Bishop 1987) but, at present, this term is commonly applied to a large number of malignancy-associated genes (Weinberg 1989) without any known relationship to viruses.
2.2.1 Groups of oncogenes
In normal cells, proto-oncogenes produce proteins that are needed for the regulation of division and differentiation. Oncogenes, implicated in the process of oncogenesis, encode proteins that may function at any level of growth regulation. Oncoproteins can be divided into two classes accoi ing to their location in the cell: those that act at the plasma membrane and in the cytoplasm, and those that act in the nucleus. Oncoproteins that function as growth factors (e.g., sis and int-2), growth factor receptors (e.g.Jms, erb-B, and neuferb-B-2),
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cytoplasmic signal transducers (e.g., abljesjps, mos, rqf, ras, and src), and transcription factors and associated proteins (p.g.,fosJun, myb, and myc), belong to signal transduction pathways which modulate gene expression and subsequent changes in cellular phenotype. In general, oncogenes influence the activity state of the signal network and, therefore, the growth properties of mammalian cells.
2.2.2 Activation of oncogenes
The activation of cellular oncogenes may be a critical step in radiation carcinogenesis. A number of oncogenes found activated in tumour cells or in cells transformed in vivo or in vitro play a central role in carcinogenesis. This has been underscored by the ability of these genes to confer a malignant state when introduced into normal cells by means of transfection (Land et ai. 1983b, Borek et al. 1987a & b, 1988).
A proto-oncogene must be changed or activated before it can produce a malignant cell. Upon encountering a growth factor, the proteins in the normal growth-regulatory pathway are transiently stimulated. In a cell containing oncogenes, however, part or all of this pathway becomes constitutively activated, enabling malignant cells to proliferate continuously (Druker et al. 1989). The endogenous cellular genes can be converted into actively transforming genes, oncogenes, by transposition and/or mutation. In general, the mechanisms by which these proto-oncogenes may be activated include spontaneous or induced point mutations, gene amplification, translocation, and retroviral (and perhaps some other DNA sequences) insertion near the proto-oncogene. Any one of these mechanisms can result in distortions of the coordinated biochemical functions of the cell.
Genomic changes, such as misregulation of genes, which lead to the transformation of a cell have been known to be caused by the insertion of a retrovirus. These viruses carry enhancer elements that increase the transcription of a neighbouring gene.
Another pathway could be via the activation of nonfunctional oncogenes captured during their passage through the chromosomes by an error-prone reverse transcription of a retrovirus. It has also been shown that the intracisternal A-panicle genes (IAP, a retroviruslike element) are responsible for mutations of the kappa-immunoglobin light chain gene in mouse hybridoma cells (Kuff et al. 1983). These particles are readily found in early mouse embryos and in the cells of many mouse tumours, but they do not have a recognized extracellular phase and have not been associated with any type of biological activity (Kuffetal. 1983).
Many mobile genetic elements (e.g., some of those present in yeast and Drosophila) are known to be transcriptionally activated by environmental stresses such as exposure to certain chemicals and UV irradiation. Contemporary movement of the LI element in somatic cells has also been observed in experimental animals; e.g., into the c-myc oncogene in some dog and rat tumours (Katziretal. 1987, Pear etal. 1988). A tumour-specific, c-myc rearrangement caused by insertion of a human LINE-J element into the second intron of
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the c-myc locus was observed in a human breast carcinoma (Morse et al. 1988). Insertion mutagenesis can also lead to the inactivation of cellular genes. The transposition of a human UNE element has been found to have inactivated the factor VIII gene in two cases of haemophilia A (Kazazian et al. 1988).
2.23 Expression of oncogenes in radiation-induced tumours
Although the specific genes that are involved in radiation carcinogenesis are only beginning to be identified, they may also include anti-oncogenes (Knudson 1985). The genetics of cancer cells thus imply that the carcinogenic effects of radiation may be mediated via the activation of oncogenes; through aneuploidy, chromosomal rearrangements, or point mutations; and/or via the homozygous inactivation or deletion of certain anti-oncogenes, such as p53 and the retinoblastoma (Rb) gene (Jenkins and Sturzbecher 1988, Friend et al. 1986).
It may be that one of the most common mechanisms in the activation of oncogenes is the translocation of chromosomal material (Klein and Klein 1984). The molecular mechanism of induction of translocations by radiation is not well understood; however, in parallel with other exchange-type aberrations (such as dicentric chromosomes), these are relatively common and caused by DNA double-strand breaks (Natarajan et al. 1980).
Radiation-induced damage to cellular DNA may result in mutations in proto-oncogenes or in the insertion of retroviral promoters and/or enhancers in aberrant positions in the host genome, thus allowing for the expression of transforming activity. This hypothesis was first tested on radiation-transformed C3H 10TI2 cells (Kirschmeier et al. 1982), but was not confirmed. Similarly, Boccara et al. (1983) did not find any evidence for linking viral long terminal repeat (U5) sequences of host origin to radiation-induced leukaemia.
As already implied, important molecular mechanisms in tumour induction and progression by radiation may involve molecular mechanisms that are not immediately obvious and that do not conform to classic phenomena such as point mutations. Radiation may activate the transcription of cellular genes that are not necessarily recognized as oncogenes but may nevertheless play an important role in the multistep process of carcinogenesis. The retroelements containing reverse transcriptase and/or integrase-related sequences, possibly enabling them to initiate their own retrotransposition, are considered to be mobile genetic elements with the potential to act as causative agents of disease. It appears that the potential impact of transpositions is not yet fully understood in association with the different types of radiation-induced hazards, including carcinogenesis and cell death.
Endogenous retroviruses and retroviral elements have been detected in the DNA of primates. Endogenous retroviral sequences resemble either full-length or truncated proviruscs in structure. Although the genomes of proviruses have generally been found to be defective, they represent a reservoir of viral genes which may be activated spontaneously by recombination events or by radiation and chemical agents. In general, endogenous
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retroviruses have long been known to be activated by radiation in mammalian cells (Duptan 1977, Schmidt etal. 1985).
Although there is growing evidence that retrotransposons can no longer be considered as silent genes which have lost their biological relevance during evolution, their actual contribution to biological processes and their possible involvement in human neoplasia demand further investigation. This topic is dealt with in more detail in the next chapter. 2 J Mobile genetic elements
The mammahan genome contains several different sequence families which represent transposable or retroposable elements. Mobile retroposable members of these families are thought to move via RNA intermediates. This process has been termed "retroposiuon" and sequences moved by this process are called retroposons (Weiner et al. 1986). There are at least four types of retroposons generated from RNA intermediates by a common mechanism involving reverse transcription and the subsequent integration of cDNAs into the (human) genome (Rogers 1985). These are the sequences similar to endogenous retroviruses, processed pseudogenes, Alu-repeated sequences (SINEs), and long interspersed repeated sequences (LINEs).
Retroposons can be divided into viral and nonviral superfamilies based on common structural features. "Retrotransposon" refers to elements which have LTRs and, thus, are more similar to retroviruses. LI elements are distinct from both retroviral-like transposons and the transposons that are terminated by inverted repeat sequences. Structural investigations have revealed two basic types of interspersed repetitive families, termed LINEs (long interspersed nuclear elements) or SINEs (short interspersed nuclear elements) by Singer (1982,1990). Retroposition is responsible for the amplification of the repetitive sequences, Alu and LI, and for the formation of processed pseudogenes. It is possible that the reverse transcriptase needed for these events is provided by the translation of an LI transcript, rather than through the translation of reverse transcriptase from endogenous retroviruses.
Many intespersed repetitive sequences were originally named according to the characteristic discrete bands seen above the continuum of single-copy fragments in a restriction digest of total genomic DNA; e.g,, the Kpn and Alu repeats (Singer et al. 1983, Houck et al. 1979). Such discrete bands reflect the presence of restriction sites at specific locations within the majority of the members of a repeat family.
2.3.1 Structure and function of LINE (LI)
Important features of the mammalian LI elements distinguish LINEs from other types of mobile genetic elements such as retroviruses, retrotransposons, and prokaryolic transposons (Table 2). Mammalian LI families share several structural features that appear to be
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Table 2. Characteristics of mammalian LINE.
1. 10 to Hr copies per haploid genome 2. lengths of 6 to 7 kb for complete elements 3. frequent deletion of a variable-length segment corresponding toth e 5' end of the LI element (5* truncation phenomenon) 4. an adenine-rich 3* terminus 5. short direct repeals (SDRs, usually < 20 bp) flanking the element 6. open reading frames (ORFs) within the element
pertinent to ZJ DNA transposition and amplification. LI contains two large conserved open reading frames (ORFs). ORF1 is about 375 codons and ORF2 about 1300 codons long, totalling about 5 kb in length (D'Ambrosioetal. 1986,Hattorietal. 1986,Loebetal. 1986). The DNA sequence of the protein-encoding portion of the rat and mouse elements is 75-95% similar. This implies that the encoded proteins are or were recently necessary for LI function. The consensus LI structure has a poly(A) tail (up to 50 bp in length) at one end and is flanked by short direct repeats, generally 5 to 15 bp long (Voliva et al. 1983). In many cases, the A-rich sequence is preceeded by the consensus polyadenylation signal, AATAAA. LI does not have long terminal repeats (LTRs). In the mouse genome, the 3* end is present at a fivefold higher copy number than the 5' end, due to the truncation of Li elements (Voliva et al. 1983).
The second ORF, ORF2, potentially encodes a polypeptide whose sequence presents homologies with reverse transcriptases and nucleic acid-binding proteins (Fanning and Singer 1987). The left end of LI families both resembles certain eukaryotic regulatory sequences (D'Ambrosio et al. 1986; Hattori et al. 1985; Furano et al. 1988; Padgett et al. 1988) and is properly oriented to regulate the transcription of the ORFs.
Hypotheses concerning die roles of LINEs range from those in which they are the main agents of regulation to those in which they are simply neutral hitchhikers without effect on the host phenotype. Heterogeneous LI transcripts are found in nuclear RNA but full-length, 'plus strand', cytoplasmic LI RNA is rare and present only in a few cell types, such as the 6.5-kb transcript seen in the human teratocarcinomacell line (Skowronski and Singer 1985) and the 8-kb RNA seen in a mouse lymphoid cell line (Dudley 1987).
2.3.2 Reverse transcriptase
Reverse transcriptase, the enzyme that can produce DNA from an RNA template, is usually thought of as the cizyme that allows a retrovirus to replicate. In recent years, reverse transcriptase has unexpectedly been found in a wide range of other genetic elements, from the widespread cukaryotic retrotransposons to mitochondrial plasmids. An unidentified reverse transcriptase has been implicated in the generation of the processed pseudogenes
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commonly found in the genomes of most eukaryotes. As already suggested, this enzyme may arise from the translation of ORF2 in the LI element
In the basal state, a small amount of reverse transcriptase activity was present in pluripotent human embryonal carcinoma Ntera-2D1 cells. Recently, it has been observed that a 2- to 3-fold increase in reverse transcriptase activity occurred following a UV dose of 20 J/m2 (Deragonetal. 1990).
2.33 RatLISE(URn)
The LI family in the rat has 40,000 members per haploid genome and it comprises about 10% of the total DNA. The LI family has probably been amplified recently since it is quite homogeneous, and most of its members are of full length (6.7 kb) (D'Ambrosio et al. 1986). Furthermore, members of the rat LI family are apparently still mobile in present-day rats since their presence or absence has been found to cause allelic variation at a number of genetic loci (Economou-Pachnis et a). 1985, Lakshmikumaran et al. 198S).
Several truncated or partial elements and one full-lengdi element have been sequenced in the rat (D'Ambrosio et al. 1986). The 5* truncation is reported to be less frequent in the rat than in mouse or human elements, although different types of experiments were performed with each species. Even though open reading frames are present in rat LJ, they are not exactly defined because of apparent chain-termination and frameshift mutations in the examples sequenced.
The left end of a typical mammalian LI family member contains a G+C richsequenc e that, in some respects, resembles the promoter of certain housekeeping and viral genes. Very recently, an internal promoterlike region of rat LI members has been reported (Furano et al. 1988). The 600-bp promoter region of (kteLIRn is active compared to the SV40 promoter in stimulating the activity of the bacterial chloramphenicol acyl transferase (CAT) gene in transient expression assays using a number of different cell lines (Nur et al. 1988). An internal RNA polymerase II promoter was also demonstrated in the human LINE within the approximately 700-bp segment immediately 5' to the apparent ATG of ORF1 (Swergold 1990).
2.4 Programmed cell death (apoptosis)
Cell death can occur via either of two morphologically and biochemically distinct modes, necrosis or apoptosis (Kerr et al. 1972, Wyllie et al. 1980, Searle et al. 1982), Necrosis is known to result from major insults or perturbations to the cellular environment. Apoptosis, on the other hand, is a naturally occurring, nonpathological form of cell death. This physiologically programmed cell death is an important phenomenon which plays a complementary role to mitosis in the regulation of cell populations. It is biochemically distinct from necrosis, the pathological mode of cell death. It has been described as a
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phenomenon whereby an endogenous suicide mechanism is activated, leading to cell death in an ordered sequence of events (Kerr el al. 1972,1987).
Cell death by apoptosis occurs under a variety of normal physiological circumstances, such as embryogenesis, metamorphosis, and the turnover of cells in normal tissues (Wyllie et al. 1980). Apoptotic cells are almost always present in growing tumours, but the significance of these observations is largely unknown at present (Moore 1987). The events surrounding the initiation of apoptosis, or programmed cell death, are obscure and remain to be elucidated. Glucocorticoid hormones, cytotoxic T lymphocytes, and natural killer cells all activate this process in target cells (Wyllie et al. 1980). It is clear that cell death by this mechanism is important under a number of physiological circumstances, including those with a direct bearing on our understanding of tumour biology.
The death of cells undergoing apoptosis is associated with such morphologic changes as the condensation of chromatin and cytoplasm, an extensive blebbing of the cell surface and a transient increase in buoyant density, and a breaking up of the cell into a number of membrane-bound fragments (apoptotic bodies). These bodies are then either phagocytosed by neighbouring cells or shed into a lumen (Wyllie 1987).
A key element of apoptotic cell death in all of the systems thus far investigated is believed to be the activation of endogenous endonuclease(s). The action of an endonuclease results in the fragmentation of the DNA into integer multiples of about 200 base pairs, thus corresponding to the approximate length of a DNA wound around a histone octamer. A specific endonuclease activation during apoptosis, resulting in the production of oligonucleosome-length DNA fragments can be resolved by agarose gel electrophoresis.
Radiation damage is known to be a potent agent for the induction of apoptosis in a number of cell types, most notably thymocytes. Lymphocytes are known to be one of the most sensitive cells in the body to low doses of radiation and are unusual in that they are killed during the interphase whereas most other cells are more susceptible to the lethal effects of radiation when they are actively dividing (Cohen et al. 1985). Lymphocyte death appears to occur via apoptosis, with rapid cleavage of DNA into the characteristic multiples of 200-base-pair units, indicating that low levels of irradiation are capable of activating an intracellular endonuclease by an unknown mechanism.
U V radiation is also known to be a potent agent for the induction of programmed cell death in human skin (Weedon et al. 1979, Hollis and Schreibner 1988). These cells were previously called dyskeratotic bodies, or sunburn cells (Wilgram et al. 1970, Olson and Everett 1975). The mechanistic aspects of radiation-induced apotosis, particularly in mammalian systems, remain poorly defined.
Previous studies indicated that a Ca2+- and Mg2+-dependent endonuclease which acts between nucleosomes is responsible for the DNA degradation (Duke et al. 1983). For the induction of cell death, an influx of extracellular calcium seems to be essential. It has been
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shown that an early sustained increase in cytosolic Car concentration was critically involved in the activation of DNA fragmentation in glucocorticoid-treated thymocytes (Truneh et al. 1985). A protein synthesized in response to glucocorticoid stimulation appeared to elevate the cytosolic Ca2* level in thymocytes by facilitating the influx of extracellular Ca2* (Cohen and Duke 1984).
2.5 Hot particles
A hot particle is somewhat arbitrarily considered to be a discrete radioactive fragment that is insoluble in water and is not larger than approximately 1 mm in any dimension (NRCP 1990). Hot particles include those emitting alpha radiation and beta or beta-gamma radiation, the latter being of concern in the nuclear reactor industry. Hot particles have also been found in the fallout of nuclear weapon tests.
For particles emitting alpha radiation, the radiation penetrates only a short distance, typically less than one hundred microns. The greatest density of ionisation occurs at the end of the alpha track. An alpha-energy-emitting particle causes a very high radiation dose around the source, but the dose falls to zero rather abruptly. A cell (nucleus) hit by a single alpha particle is severely damaged and the survival probability is about 50%. It is important to realize that an alpha source does not create a dose gradient around it and, henc?, a direct comparison of alpha-emitting hot particles with those used in the present study is not warranted.
Hot particles in the nuclear power industry most commonly contain neutron activation products, such as "to, or fission products. The likely source of the fairly large particles (diameter commonly over 100 \un) containing *°Co is particles of wear-resistant alloy, e.g., from valve seats, which contain a high percentage of stable cobalt and become activated in the core. The radioactivity of the wCo particles ranges from 40 Bq to 20 MBq (most commonly from 400 Bq to 200 kBq). The source of the particles which contain fission products is fuel elements which have defects in their cladding. The radioactivity of the particles containing fission products ranges from 40 Bq to 400 kBq (most commonly from 400 Bq to 40 kBq; Wamock et al. 1987). These particles should, in principle, be similar to the hot panicles released from die Chernobyl reactor (see page 31) and were used in the present study.
Hot panicles in fallout of nuclear weapon tests were very small (mean maximum diameter about 4 |im), but the activity of these particles was low (up to about 100 Bq), Gammaspectometric analyses have revealed the following components in most of the particles examined: 95Zr/Nb, wMo, ,3,I, l32Te/I, ,40Ba/La, 14,Ce, ,44Ce, and ,47Nd (Rajewsky et al. 1962).
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3 AIMS OF THE STUDY
The experiments included in mis study were carried out for the following purposes:
1. To isolate and identify Chernobyl-released hot particles. 2. To develop experimental assays regarding the effects of these particles, especially on the induction of malignant transformation. 3. To study the activation of oncogenes in radiation-induced transformation. 4. To clarify the mechanisms which lead to "spontaneously" initiated programmed cell death in rat chloroleukaemia. 5. To study the premature activation of this suicidal mechanism by UV and ionizing radiation.
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4 MATERIAL AND METHODS
4.1 Cell culture
The C3H10T112 mouse embryo fibroblasts (clone 8), developed by Reznikoff et al. (1973a, b), were used in the present transformation studies (IIJII.IV). Only cells from passage 8 through 14 were used in die experiments. The cultures were grown in Eagle's basal medium (BME, Gibco), supplemented with 10% heat-inactivated fetal calf serum (Gibco) and antibiotics (penicillin, 100 units/ml, and streptomycin, 100 iig/ml). Cells were maintained at 37°C in a humidified, 5% CO2 atmosphere and were passaged once or twice weekly.
Rat chloroleukaemia, induced by gascric instillation of methylcholanthrene (Shay et al. 19SS), has been maintained in our laboratory by suspension culture during the last 12 years. The cells are grown in different types of culture media, commonly Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 percent foetal calf serum, penicillin (100 units/ml), and streptomycin (100 Mg/ml). The cultures were incubated at 37°C in humidified air containing 5 percent CO2 (V.VI.VII).
4.2 Cell morphology
Chloroma cell morphology was evaluated on May-Grilnwald-Giemsa-stained cytocentrifuge cell preparations (Fig.VI-1, Fig.VII-4). Apoptotic cells were identified using the criteria given by Kerr et al. (1972) and Wyllie et al. (1980). 4.3 Irradiation
4.3.1 X-irradiation
X-irradiation was delivered from a Valmet BR1001 three-phase generator (W anode X-ray tube, 110 kV tube voltage with an added filtration of 1.7 mmAl). Chloroma cells grown in plastic culture petri dishes (dia. 90 mm) were irradiated at room temperature at a dose rate of0.2Gy/min(Vll).
4.3.2 Irradiation by Co gamma rays
Semiconfluent cultures were irradiated with gamma rr.ys at room temperature, fed by fresh culture medium, and subcultured at 400 viable cells per culture. The dose was 3 Gy and the dose rate 0.4 Gy/min (II).
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4.3 J UV irradiation of chloroma cells
The cultures were exposed from below to a UV transilluminator source (Fotodyne Inc.) at room temperature for varying time periods. All UV exposures were conducted in polystyrene 90 mm culture flasks (Falcon). Cells were UV-irradiated at room temperature and all cell cultures were then further incubated at 37°C in a humidified 5% (Xh/95% air atmosphere (VII). Irradiance was measured (Optronic Spektroradiometer, Model 742) at distance of 35 cm from the table surface, at 24°C (VII).
4.3.4 Hot particles
Hot particles are tiny fragments of authentic nuclear fuel released from Chernobyl or artificial particles produced by bombarding uranium oxide (UO2) with slow neutrons. The authentic particles have been isolated from environmental samples collected in Finland and Kiev (I-IV). Hot particles were sterilized by autoclaving and then added to growing C3H 10TJ/2 cultures (25 cm2 Falcon tissue culture flasks).
4 4 Gammaspectrometric analyses (I-IV)
Hot particles were analysed for fallout, gamma-emitting nuclides. Gammaspectrometric analyses were performed using either lithium-drifted or high purity detectors with relative efficiencies of between 15% and 39%. The activity contents of the samples were calculated using the computer programme GAMMA-83 (Sinkko and Aaltonen 1985).
4.5 Plasmids and probes
Fragments of LIRn cloned from chloroma cells (VI) were used throughout this study. The main probe used here was a 5.5-kb BamHI fragment of chloroma LIRn (pCB4), covering the two reading frames of the element almost in full. The cellular and viral oncogene probes used are shown in Table 3.
Each probe was labelled with 32P-dCTP (Amersham) using a multiprime DNA labelling or nick translation system according to the supplier's instructions (Amersham).
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Table 3. Oncogene probes
Oncogene Fragment Length Source Category used
v-sis Sstl-Xbal 983 bp Woolly growth factor Monkey
v-erb-B Sstl-PvuII 1.700 bp chicken transmembrane growth y-fms BamHI-EcoRl 500 bp cat factor receptors
y-abl Hindll 1,740 bp mouse membrane-associated v-src EcoRI 2,950 bp chicken tyrosine kinases v-fes PstI 460 bp cat v-fes PstI 450 bp cat v-fps PvuII-Smal 1,300 bp cat
v-Ha-ras SsU-PstI 730 bp rat membrane-associated c-Ha-ras human guanine nucleotide v-Ki-ras SsUI-HincII 618 bp rat binding proteins c-Ki-ras EcoRI-Hindlll 640 bp human N-ras human
v-rqf XhoI-SstI 300 bp mouse cytoplasmic serine- threonine kinases
v-erb-A PstI 478 bp chicken cytoplasmic hormone v-erb-A Psa 450 bp chicken receptors
v-myb BamHl 7,200 bp chicken nuclear factors Q-myc Clal-EcoRI 1,800 bp human c-myc PstI 400 bp human N-myc EcoRI-BamHI 1,000 op human v-fos Pstl-PvuII 1,000 bp mouse
4.6 Nucleic acid analyses
High-molecular-weight nuclear DNA was isolated by proteinase K digestion and organic solvent extraction. The transformation ofEscherichiacoli, restriction enzyme digests, DNA fragment ligations, and fragment isolations were done using standard methods (Maniatis et al. 1982, Ausubelctal. 1987).
10-20 \xg of total DNA was digested with restrictionendonuclease s and fractionated by electrophoresis through agarose gels. After electrophoresis, the DNA was iran&fened to
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nitrocellulose paper in lOxSSC and fixedb y UV irradiation or by baking. DNA fragment sizes were calculated using X-phage DNA cleaved with the restriction endonucleases, EcoRI and Hindin, as a standard (Boehringer-Mannheim). Dot or slot blots were performed with a filtration device (Minifold I or II, Schleicher and Schuell) using nitrocellulose filters pretreated with lOxSSC (DNA) or 20xSSC (RNA). The nitrocellulose membranes were hybridized with a probe which was labelled by nick translation or random priming with 32P(1 to 2x10s cpm/ng).
Total cytoplasmic RNA was isolated by cell lysis in cold Nonidet P-40 (BDH) buffer, followed by phenol extraction and ethanol precipitation. The RNA was analysed by direct slot or dot blotting of the RNA onto nitrocellulose. Polyadenylated RNA was isolated by oIigo(dT) chromatography from cell lysates. Aliquots of RNA were electrophoresed in 1.0% formaldehyde-agarose gels, transferred to nitrocellulose filters in 20xSSC, and fixed as above.
The hybridizations and washings were performed under stringent conditions for eukaryotic probes (hybridization: 50% formamide, 3xSSC, lxDcnhardt's solution, 50 mM HEPES, 150 ug/mlofcarrierDNAat42oC;lastwash:0.1xSSC,0.1%SDSat55°C) and less stringent conditions for all other probes (hybridization: 40% formamide, 3xSSC, IxDenhardt's solution, 50 mM HEPES, 150 u\g/ml of carrier DNA at42°C; last wash: lxSSC, 0.1% SDS at 50°C). After washing, the filter was exposed to X-ray film at -70°C or at room temperature for 1-10 days in the presence of intensifying screens. Autoradiograms were developed according to an automatic standard technique (Sakura QX).
4.7 DNA fragmentation studies
The assay described here reveals the biochemical indicator of apoptosis (i.e., the characteristic DNA degradation pattern accompanying apoptosis) not studied in the published papers (VI-VII). Cell pellets (2-4xl07 cells) were resuspended in 50 \si of PBS, followed by 2 ml of a digestion solution (10 mM EDTA, 50 mM Tris, pH 8) also containing 0.5% sodium lauryl sarcosine, 100 u,g ml1 proteinase K, and 200 p.g ml'1 RNase A. Digestion was carried out at 50°C for 1-3 h. The crude DNA preparations were extracted with an equal volume of phenol, followed by two chloroform/isoamyl alcohol extractions. DNA preparations were then brought to 2.5 volumes by the addition of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) and centrifuged at 13,000g for 10 min to separate the intact chromatin from the fragmented. The supematants, containing fragmented DNA, were placed in separate tubes and the DNA was precipitated with 2.5 volumes ethanol at -70°C. The DNA precipitates were recovered by centrifugation at 13,000# for 10 min, dried, and resuspended in TE buffer. The DNA concentration was calculated by determining the OD at 260 nm. Electrophoresis of the fragmented DNA was carried out in 1 % agarose gels at 2 V per cm. A Hindlll and BamHI digest of XDNA was applied to each gel to provide a molecular weight standard. After the completion of electrophoresis, the gel was soaked in buffer containing 1 jig/ml cthidium bromide and then photographed.
29 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
4.8 In vitro transformation assays
C3H10T1/2 cells were grown at least two days before irradiation was begun. This allowed the cells to regain their exponential gtowth before exposure. During irradiation, cells were sealed in plastic flasks filled with prewarmed media. Immediately after inadiation, the cells were dispersed and plated into multiple, 25 cm2, tissue culture flasks at a density such that 200-400 colonies developed in each flask. The growth medium was renewed weekly.
For transformation assays with hot particles, a single uranium particle was added to each test culture.
4.9 Scanning electron microscopy
Cell cultures in Falcon flasks were fixed in situ by gently decanting the growth medium and fixing the cells immediately with 2-3% glutaraldehyde in 0.1 M phosphate buffer at room temperature. After 1 h and three buffer washes, the material was post-fixed in 1% Os04 in the same buffer for 1 1/2 hours at room temperature. For SEM, the flasks were filled with distilled water after three further buffer washes and areas selected under the inverted microscope were cut out with z wanned scalpel. These discs of plastic were then dehydrated in ethanol, critical point-dried (Balzers Union, CPD-020), and sputter-coated with 20 nm of gold (JEOL FINE COAT JFC-1100 sputtering device) before examination in a JEOL JSM-35C SEM (JEOL LTD, Japan).
Hot particles were sputter-coated with a 20 nm gold layer before scanning electron microscopy, and analysed by SEM and a microprobe X-ray analyser. Elements from Na to U can be characterised in the X-ray microanalysis (PGT System III EDS Microanalyzer, Princeton Gamma-Tech Inc., USA).
4.10 Time-lapse videography
The development of the particle-associated wound and the growth of a transformed focus were followed by time-lapse videography. The films from growing cultures at 37°C were taken with a time-lapse factor of 160, documenting 3 weeks onto a 3-h casette. These films complement the studies reported in the published papers (III—IV).
30 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
5 RESULTS AND DISCUSSION
In the present overview, only a few aspects of the published papers (I-VII) are considered in detail. The selection of these aspects reflects the potential importance of the findings that I attach to them at present. 5.1 Hot particle characteristics (I-IV)
Hot particles with beta-emitting radionuclides were detected in the fallout originating from the Chernobyl reactor; they varied considerably in radionuclide composition and activity. The term "hot" comes from the rapid blackening of an X-ray film exposed to the radioactive particle. Particles used in the present studies produced a detectable signal within seconds.
Uranium aerosols transported by air to distant areas (e.g. Kiev) and hot particles isolated from a contaminated Russian train were used in this study. After the Chernobyl accident, up to 10,000 hot particles per square meter of ground surface were detected in some areas in Finland (MI). The hot particles were first located by autoradiographic techniques and, after isolation, particle characteristics were analysed by SEM, X-ray microprobe, and gamma spectrometric techniques.
The particles contained nonvolatile fission products including the B-cmitters, 95Zr, 103Ru, I06Ru, 141Ce, and 144Ce (Tables 1-1,11-1, III-l). Aerosols always contain zirconium and cerium in quantities that are proportional to the fuel burnup. The observed ruthenium activity varied strongly between particles, suggesting different aerosol formation mechanisms (II). Ruthenium was oxidized to R11O2 or, possibly, to ruthenium carbonate in the graphite fire. Ruthenium was often detected in aerosols that were smaller in size than die direct fuel fragments; apparently, oxidized volatile ruthenium escaped from the uranium matrix and condensed later on other tiny fragments of the reactor fuel (II). These highly active aerosols can be transported more easily in air.
Isolated samples were subjected to scanning electron microscopy (SEM). The scanning electron micrographs of hot particles are shown in Figs. H-2, Ii-8, IIM, and IV-3. X-ray spectra from the hot particles indicated that the particles were chemically pure uranium; i.e., they consisted of fragments of nuclear fuel (Fig. II-3).
These hot particles may pose a potential health hazard to humans distinct from the acute B-bums of skin and mucous membranes. One obvious hazard is the long-term retention of a relatively small particle in the respiratory tract or in the lung-associated lymph nodes. The risk from inhalation of Ru-enriched hot particles might exceed that from all other exposure pathways of die Chernobyl fallout. For these reasons, the biological effects of the hot particles were studied in some detail.
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5.2 Biological effects of radiation 5.2.1 Malignant transformation
The C3H10T1I2 mouse fibroblast cell line, commonly used in radiation transformation assays, showed malignantly transformed foci at a frequency of 2.5x1c4 per surviving cell after a dose of 1 Gy of external gamma radiation (III)- This transformation frequency is consistent with several other studies (Hill et at. 1987, Terzaghi and Little 1976, Han and Elkind 1979, Miller and Hall 1978, Little 1979).
The Chernobyl-released and artificially activated uranium particles were extremely effective in producing premalignant cellular changes and expression of malignant transformation in vitro (II,III,IV). Different fission products of an immobilized uranium particle create a strong dose gradient around it, delivering a lethal radiation dose to adjacent cells and a high, but sublethal, dose to the surrounding cells up to a distance of several mm or, depending on the particle activity, even a few cm. Transformed foci were seen in alt cultures in 4-8 weeks and all foci were within 2-4 mm of the radiation source (HIJV).
The transformation of cells by the particles was not associated with the radiation dose in the conventional sense. In terms of an average cell dose over the whole population, the point source is orders of magnitude more effective than an evenly distributed gamma radiation. In terms of an effective dose equivalent as defined by the ICRP 26 (1977), no malignant transformation should have been seen in any of the test cultures. Just the opposite was seen, however, and cell transformation occurred in every single culture (III, IV).
Apparently, the major factor responsible for this effect is the focus promotion caused by a wound-mediated, permanent increase in cell proliferation among the population receiving a high, but sublethal, radiation dose. The hard beta activity of the particle must, however, be high enough lo result in a dose rate on the order of one gray per hour or more adjacent to the particle; hence, to kill enough cells and to induce a sufficiently large chronic wound around the particle. An apparent threshold activity seems to be at least a few hundred Bq of 144Ce or 106Ru (further details not shown in the papers of this thesis). In the case of hard beta emitters, such as the daughter nuclides of IMRu and 144Ce (rhodium and promethium), the range of the beta particles was sufficient (several mm) to cause a high, but sublethal, dose to a sufficiently large number of rapidly proliferating cells. These conclusions were directly confirmed through the vidcofilms showing the sustained high mitotic activity and locating at an early stage the places of transformation. An apparent fat transfonnation of the C31110T1I2 cells was also common in the radiation field, but these cells did not seem lo progress to malignant foci. Some videoprints are shown in Figs. 1 and 2; nose results arc not given in (he accompanying papers (H-IV).
32 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
Fig. 1. Permanent "wound" in the C3H10TII2 cell layer, induced by an artificial hot particle (black spot in the right lower corner). Phagocytosis of uranium miniparticles can be seen at the upper edge of the wound. Videoprintfrom a follow-up culture.
Around the death zone, the cells receive the highest possible sublethal dose and, therefore, the probability for malignant transformation (initiation) was high (II-IV). The indirectly induced and sustained proliferation of these cells then results in a rapid fixation of the genomic change and, thus, promotes its expression and clonal development This result is strongly reminiscent of the situation where a similar effect is obtained by subculturing. If cultured C3H10TJ/2 cells are irradiated with a high acute X-ray dose, allowed to grow to confluence, and then maintained for 4-5 weeks, a dose-dependent number of transformed malignant foci arises (II-IV). In this type of assay, transformed foci are expressed, per culture, at a frequency that seems to be constant; he,, they are independent of the number of irradiated cells, at least over a certain range of cell densities. However, if the irradiated cells arc subcultured (e.g., at 10^ dilution) after reaching confluence and then allowed to regrow to confluence, the same constant number of foci develops on each new plate. Thus, the risk (frequency of expression of malignancy) is increased 10,000-fold simply by subculluring (Kennedy et al. 1984).
33 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
•¥' * JPIH^^^H * * '%.**3pMBH * ^ « * * ,Ät < vP
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- 1 ' ' '..-•>« <£3.-(K>'Sö; "3HJ *:'' *IV**> *i.x» ;l#i:S£4« ' .*• - '"**''/. *'v
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f/g. 2. Far transformation ofC3H)'07T/2 cells near the wound border, induced by un artificial hot panicle. Videoprintsfrom a follow-up culture.
34 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
5.2.2 Radiation-induced wounds and the role of mitogenesis
Time-lapse videography has shown that noniransfonned C3H 1CTJI2 cells continue to divide after reaching confluence (not shown in the accompanying papers). However, cells which cannot attach to a solid surface die (apoptosis) and the cell multilayer does not form. Later, mitotic activity also stops. Radiation-transformed cells do not die and daughter cells, after mitosis, spread over the underlying cell sheet, forming a thick cell colony. The first visible sign of a malignant focus appears as a criss-cross architecture of the cell layer. Initially, the net growth of the focus is prevented by the death of cells that do not find a solid surface immediately after mitosis. When the anchorage dependence disappears, fast growth of the focus ensues. Growth occurs in relatively short, intensive bursts of mitoses following medium change. A thick, transformed focus is very different from a confluent, nonmitotic C3H10T1I2 cell layer (Til JV).
The formation of a wound is necessary to stimulate the surrounding cells into sustained proliferation and, hence, toward a rapid and effective "fixation" and multiplication of a harmful genomic change which is induced in the target cells. In order to observe die cellular changes that took place during die irradiation more closely, several cultures were followed by time-lapse videography. In a nonconfluent culture containing a hot particle, C3H 10Tlt2 cells divided rapidly. A confluent monolayer was first formed, achieving close contact with die hot particle. No cytotoxic effects have been seen in these cells, even very close to a hot particle, because die cells proliferated rapidly in a nonconfluent culture before reaching a lethal radiation dose. From die artificial hot particles, however, some uranium was released and was phagocytized by nearby cells (Fig. 1). These cells did not die rapidly, but they did not proliferate either.
A dividing cell is much more at riskfo r mutation than a quiescent cell. The time interval for DNA repair during cell division is short. Single-stranded DNA is without base-pairing and histones and, thus, is more sensitive to damage than double-stranded DNA during cell division. Cell division triggers mitotic recombination, geneconversion, and nondisjunction, which together seem orders of magnitude more effective dian an independent second mutation. Cell division allows gene duplication which can increase the expression of oncogenes that are otherwise weakly expressed (Orr-Weaver et al. 1986); for instance, it can increase die expression of die myc and fos oncogenes (Coni et al. 1990). Cell division also allows 5-methyI-cytosine in DNA to be lost which can result in dedifferentiation (Wilson et al. 1987, Lu et al. 1988), thus often causing further mitogenesis.
5.2.3 Oncogenes
The transformed foci were isolated, cloned, expanded, and analysed for die activation of different oncogenes at die transcriptional level. It appears from die results shown in Table 4 (and in Table 111-2) that almost all of the tested genes may be activated by radiation, but for none of diem is the change frequent It therefore seems that Uiese proto-oncogenes may not be causally associated with die transformation or at least do not represent a specific
35 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
target for a radiation-induced change. As with acute, large-dose, gamma radiation, hot-particle-transformed cells revealed variably enhanced transcription of several oncogenes.
One possibility for a common change, however, remains. It seems that the mobile genetic element LINE is commonly activated by radiation (VII). It may also be worth noting here that the oncogene, Blym (Cooper and Neiman 1980), is actually a 590-bp fragment from LINE. The Blym gene was initially believed to be a fragment of chicken lymphoma DNA which produced, by transfecuon, oncogenic transformation in mouse fibroblast cells (Cooper and Neiman 1980). It is not evident, however, how \hisIJNE fragment of the mouse genome could have a phenotypic effect on mouse cells (Rogers 1986). The mouse genome already contains thousands of copies of such sequences. I suggest that nonlethal remodelling of the genome by integration of LINE elements in critical positions in the DNA may be responsible for many of the changes seen in the irradiated cells. If the radiation-induced mobility of LINE is extensive, the genome is destroyed and the cells die (VI-VII). In some respects, LINE activation resembles the radiation-induced activation of proviruses.
Table 4. Summary of oncogene transcription in malignantly transformed cell lines compared with nontransformed C3H10TH2 cells.
Probe Number of foci Increased Total signal * tested
c-myc 1 7 \-myb 3 8 s-fos 0 5 c-Ha-ras 2 11 c-Ki-ras 1 10 y-erb-A 2 7 v-erb-B 3 4 \~fms 2 5 \-abl 4 8 v-sis 1 11 v-raf 0 6
* Two-fold or more, compared with non (raniformcd C3H 10TU2 cell*.
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The identification of specific oncogenes (and anti-oncogenes) that may be involved in radiation carcinogenesis is still a largely unexplored area. However, the Ki-ras oncogene was reported by Guerrero et al. (1984) to be activated by a point mutation in the majority of cases of thymic lymphomas induced by irradiation in C57B1 mice, in contrast to the N-ras oncogene which was activated in the majority of such neoplasms induced by 4-nitrosourea. Similarly, the Ki-ras oncogene was found to be activated in a large percentage of rat skin carcinomas induced by ionizing irradiation, along with the activation, gene amplification, and enhanced expression of the myc oncogene (Garte et al. 1986). Each of a number of primary radiation-inducedlun g tumours examined expressed c-myc and one or more c-ras oncogenes at levels 2-to 4-fold higher than the beta-actin gene, which was used as a benchmark (Kelly et al. 1989). Enhanced expression of the oncogene, c-myc, has also been seen in radiation-transformed C3H 10T1/2 cells (Billings et al. 1987) and in human WIS cells (Mizuki etal. 1985).
In view of the evidence that more than one oncogene must be activated to transform a cell, it is not clear how the ras and myc oncogenes have contributed to the pathogenesis of the tumours, nor is it clear whether they were activated early or late in tumour induction. Nevertheless, the increased susceptibility of C3H 10T1/2 cells to radiation-induced transformation after the introduction of an exogenous c-myc oncogene has been interpreted to suggest that die activation of c-myc plays the role of the second event in a two-stage transformation sequence (Sorrentino et al. 1987).
A series of transplanted tumours and cell lines derived from radiation-induced bone tumours revealed increased RNA levels of c-Ki-ras and c-myc in several of the tumours (Schön et al. 1986). Less frequently, overexpression of die ptoto-oncogenes, c-sts, c-abl, c-bas, and c-fos was also reported by these authors, but they concluded that there was no indication that any particular proto-oncogene or combination of proto-oncogenes was associated with murine osteosarcomas. It has been shown that the oncogene, c-myc, is expressed in cells of transformed foci, but the presence of a mutated c-ras in transformed C3H10T1/2 cells has not been established (Sawey etal. 1987). A unique, but so-far unidentified, transforming oncogene has been demonstrated in cell lines derived from foci generated by radiation in mouse C3H10T1I2 and hamster embryo cells (Borek 1987b).
Many of the positive reports of oncogene activation derive from transfection studies with DNA isolated from malignant cell lines. This technique was used, e.g., in studies involving thymic lymphomas induced by y-radiation in the mouse. It was observed that a point mutation in the c-Ki-ras proto-oncogene, leading to the subsequent change of one amino acid in the translation product, was responsible for tumour development (Guerrero et al 1984). Point mutations in the c- Ki-ras and N-ros proto-oncogenes have also been detected in other rodent tumours induced by ionizing radiation (Sawey etal. 1987,1989; Diamond et al. 1988). The mutations that occur in different tumours are not necessarily identical, implying a modifying cellular mechanism that will give rise to the heterogeneity.
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The amplification of c-myc was found in 12 out of S3 murine osteosarcomas induced by radiation. The expression of c-myc RNA was increased 6- to 50-fold in these tumours which harboured multiple gene copies of c-myc. The amount of c-myc RNA roughly correlated with the copy numbers of the amplified c-myc genes (Sturm et al. 1990). These results suggest that the c-myc gene is involved in the multistep process of osteosarcomagenesis and the differentiation process of osteogenic cells. Amplification of the c-myc oncogene has also been demonstrated by other authors in radiation-induced osteogenic sarcomas (van der Rauwelaert et al. 1988). The erb-B oncogene was amplified in two out of ten primary radiation-induced lung tumours examined (Kelly ct al. 1989). In the present experiments, amplification of the c-myc oncogene in the C3H10T1I2 cells was seen in one "spontaneously" transformed focus (obtained by extensive passage of the cells), but apparently in none of the radiation-transformed foci (111).
Until more is known about the identity, regulation, and function of the oncogenes (and anti-oncogenes) that are involved in radiation carcinogenesis, their true roles in die process will remain uncertain. One interesting possibility, suggested before (p. 36), is the activation of a mobile genetic element by radiation and its incorporation into a critical location in the genome. Besides the activation of retroviruses by radiation (Duplan 1977, Schmidt et al. 1985), this proposal also arises from the experimental observations described below, although the endpoint screened here was apoptosis (programmed cell death).
53 Programmed cell death (apoptosis)
5.3,1 Growth and morphology of chloroma cells (V,VI,VII)
Chloroleukaemia is a transplantable myeloid leukaemia, induced in rats by gastric instillation of methylcholanthrene. These cells can also be maintained in vitro and they grow in suspension culture in different types of media, including synthetic medium supplemented with insulin and transferrin (V). Detailed kinetic and time-lapse cinematographic analyses have shown that during the exponential growth phase, all cells proliferate actively and no cells are lost through death. At a certain critical cell density, which is slightly different in different culture media, chloroleukaemia cells stop traversing the cell cycle and they appear to pile up in an early stage of the Gi phase (V-VI). Net population growth does not change for many hours, however, because cells that have passed the apparent, but so far unidentified, restriction point continue to divide normally. However, after about 11-12 hours, when the cells that have ceased to progress in the cycle should divide, entry into mitosis decreases drastically and the net population growth stops abruptly. At this stage, and actually for many hours later, all these phenomena are fully reversible; dilution of the population back to a low cell density in fresh medium restores fast exponential growth. However, a medium change alone is not sufficient; if the cells are not subcultured, the population commits group suicide, with the cells morphologically undergoing a programmed death, or apoptosis, and not death by toxic necrosis (VI).
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The morphological features of apoptosis have been well documented and include nuclear chromatin condensation, cell vacuolation, plasma membrane ruffling, and the generation of apoptotic bodies (Wyllie et al. 1980). Cells with these characteristics were seen to increase in number in cultures of chloroma cells both spontaneously and after exposure to ionizing and UV irradiatkH) (Figs. VI-1 .VII-4). These cells had highly-condensated nuclear chromatin present in numerous small nuclear fragments within the cell cytoplasm.
5.3.2 DNA fragmentation
In the original papers (VI, VII) of this thesis, the biochemical markers of apoptosis were not studied. This is not important for the conclusions reached, but it may be cnsidered as a weakness which does not justify the use of the term apoptosis for the observed cell death because this pattern of DNA degradation is considered characteristic to apoptosis (Smith etal. 1989,Shietal. 1989, Wyllie 1980, Wyllie etal. 1984). To meet this potential criticism, the formation of an oligonucleotide ladder in dying chloroteukaemia cells was studied. Fig. 3 shows that the DNA extracted frrom apoptotic cells was indeed fragmented into a ladder pattern (lane S), chracteristic of apoptosis, whereas DNA from nonapoptotic cells was not similarly cleaved (lanes 2-4).
Fig. 3. Agarose gel electrophoresis of the fragmented chloroma DNA contained in the 13,000g supernatant from lysates of apoptotic and nonapoptotic cells. DNA from UV-irradiated cells shows a clear ladder of degraded DNA bands, the sizes of which are multiples of about 200 bp at 24 h after UV-irradiation (lane 5). This ladder formation is absent in control cells (lanes 2 and 4) and in UV-irradiated cells at 4 h after irradiation (lane 3). Lane I is the molecular weight standard (a HindJII and BamHI digest ofkDNA).
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5.33 Transcriptional activation of LIRn (VI, VII)
During exponential growth of chloroleukaemia cells, LINE transcripts are present in the cell cytoplasm (V-VII). It is also worth noting that the expression of c-Ki-ras, which is almost certainly associated with the malignant character of chloroleukaemia cells, is strong at this stage (VI)- At the critical cell density, UNE is suddenly activated and the transcript level increases 5- to 10-fold without any apparent exogenous reason; at the same time, c-Ki-ras is repressed to an undetectable level. If the processes are not reversed by subcuHuring, programmed cell death (apoptosis) follows from die sudden incorporation of about 300,000 copies of the LINE element into random locations in the cell genome, apparently by retroposition via the RNA intermediate, thus creating lethal mutations (VI).
UV and ionizing radiation induce the strong transcriptional activation of UNE and the programmed death of chloroleukaemia cells prematurely at any stage during exponential growth, before die spontaneous activation at the critical cell density. Especially after UV exposure (at least UV-B), UNE activation occurs quickly, within 2 hours or less, and its transcript level increases up to 70-fold in 20 hours. Interestingly, hyperthermia does not activate LINE in chloroleukaemia cells and the cells are killed, after a severe heat treatment, by toxic necrosis rather than apoptosis (VII).
Most information concerning responses induced by nonionizing radiation in eukaryotes has been obtained using UV-C radiation at 254 nm. Nevertheless, the shortest wavelength in the solar spectrum is around 290 nm and the UV-B (290-320 nm) and particularly die U V-A (320-380 nm) regions of sunlight appear to interact with cells quite differently from UV-C radiation.
DNA synthesis (and, therefore, repair) is inducible in mammalian cells. For instance, UV-induced post-replication repair synthesis appears to be enhanced if cells are exposed to a low UV fluence several hours prior to the main UV challenge (D'Ambrosioand Setlow 1976). It is also known that UV-C irradiation of human fibroblasts leads to an immediate inhibition of DNA synthesis, followed by recovery a few hours later (Moustacchi et al. 1979). UV radiation is also able to modulate gene expression in mammalian cells. In addition to increasing the level of expression of certain cellular genes, UV radiation may be able to switch on genes which are normally quiescent. The activation of transcription seems to be associated with the extensive demethylation of one of the two copies of a gene (Lieberman et al. 1983). Since UV-B radiation induces similar types of DNA damage as U V-C (254 nm), it may be expected that it activates gene expression in a similar manner.
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6 CONCLUDING REMARKS
The Chernobyl-released uranium particles are extremely effective in producing of malignant transformation in vitro. The major factor responsible for this effect is focus promotion caused by the wound-mediated permanent increase in cell proliferation among die population receiving a high, but sublethal, radiationdos e (mitogenesis associated with mutagenesis). In other words, the indirectly induced and sustained proliferation of the cells results in a rapid fixation of a genomic change and, therefore, strongly promotes its expression and clonal selection.
In the present study, the expression of oncogenes in different radiation-induced foci was found to vary from focus to focus. Thus, any observed enhanced oncogenic expression may not be causally associated with transformation, or at least such expression may not reflect hits to a specific oncogene target in radiation-induced carcinogenesis. Numerous recent studies of oncogenes have implicated them as specific loci for the genetic alterations which earlier had been confined to gross chromosomal abnormalities and to changes in ploidy. These microscopically visibb alterations were difficult to correlate with actual biochemical events that take place in malignant cells.
UV light and ionizing radiation induce the strong transcriptional activation of a mobile DNA element (LINE, the long interspersed repetitive nuclear element). This activation of LINE is rapid and premature during the exponential growth of chloroleukaemia cells and the exogenously induced activation is followed by programmed cell death (apoptosis). It appears that the radiation-induced activation of LINE is based on, or at least associated with, direct DNA damage and it may resemble the activation of provinises by radiation.
The activation of LINE is followed by programmed cell death in a sequence of events identical to that seen in spontaneous LIRn activation. Apoptosis following radiation may also be viewed as an altruistic phenomenon eliminating potentially deleterious cells.
On the basis of the present results, it is implied that radiation-induced primary DNA damage may sometimes lead to complex molecular mechanisms via retropositions or transpositions of LINE or other mobile elements, distinct from radiation hits directly to proto-oncogenes or their regulatory sequences. If this conclusion is correct, it would alter the current opinion of radiation-induced health hazards.
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ACKNOWLEDGEMENTS
The present study was carried out in the Laboratory of Radiobiology of the Finnish Centre for Radiation and Nuclear Safety, Helsinki, Finland.
My warmest thanks are due to Professor Tapio Rytömaa, MD, DScMed, my supervisor and nearest coworker, who skilfully introduced me to scientific work. He has shared the successes and misfortunes of research with me and encouraged me to carry on. Tapio's enthusiastic guidance and wealth of ideas have been of great importance for the completion of this study.
I am also grateful to Professor Olli Halkka, PhD, the Head of the Department of Genetics, University of Helsinki, for his friendly and uncomplicated attitude toward my studies.
I wish to thank my colleagues and those who have been involved with my work, especially Dr. Harri Toivonen, DTcch, for his help and interest in this work. My most sincere thanks are due to Ms. Ulla Dcnnehy, Ms. Pia Kontturi, Mr. Teuvo Parviainen, Ms. Marja Huuskonen, and Ms. Leni Soini for all their help and excellent technical assistance that was needed throughout this work.
I am deeply grateful to Mr. Simo Lehtinen, MScTech, and Mr. Seppo Hornytzkyj, MSc of the Institute of Electron Microscopy, University of Helsinki, for their invaluable help in the use of methods of scanning electron microscopy. Special thanks are also given to Mr. Mauri Kaituri, MSc, for his help in the collection of hot particles. I also wish to thank Ms. Kirsti Leszczynski, MSc, Dr. Antti Servomaa, PhD, and Mr. Erkki Rantanen, MScTech, for their help in the irradiation of cells and in irradiation measurements. I am indebted to Mr. Kari S inkko, Lie Phil, and Ms. Hannele Aaltonen, MSc, for their help in the gamma spectrometric measurements.
I want to thank Professor Olli Halkka, PhD, and Dr. Sisko Salomaa, PhD, for critically reviewing the manuscript and for their comments. I also thank Ms. Lisa Patterson, MSc, for revising the English language, as well as for her friendly attitude and interest in my work,
I am deeply grateful to Professor Antti Vuorinen, DTech, the Director General of the Finnish Centre for Radiation and Nuclear Safety, for the privilege letting me use the excellent working facilities.
Finally, I wish to thank my family and friends for their support. My dearest thanks are due to my husband, Antti, for his unfailing support and encouragement, which were necessary for me and this work, and to our sons, Jarno and Tommi, for their patience and love although I was "invisible" during long times.
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REFERENCES
Aisenberg A.C. New genetics of Burkitl's lymphoma and other non-Hodgkin's lymphomas. Am. J. Med. 1984; 77:1083-1090.
Ausubel F.M., Brent R., Kingston R.E., Moore D JX, Seidman J.G„ Smith J.A„ Strahl K., eds. Current Protocols in Molecular Biology, New York: John Wiley & Sons, 1987.
Balmain A. Transforming ras oncogenes and multistagecarcinogenesis.Br.J. Cancer 1985; 51:1-7.
Balcer*Kubkzek E.K., Harrison G.H., Thompson B.W. Repair time for oncogenic transformation in C3H/10T1/2 cells subjected to protracted X-irradiation. InL J. Radiat. Biol. 1987; 51:219-226.
Barrett J.C, Fletcher W.F. Multistep models of carcinogenesis. In: Mechanisms of environmental carcinogenesis. Florida: CRC Press, 1987:73-116.
Baverstock K.F., Will S. Evidence for the dominance of direct excitation of DNA in the formation of strand breaks in cells following irradiation. Int. J. Radiat. Biol. 1989; 55: 563-568.
Billings P.C., Shuin T., Lillehaug J., Miura T., Roy-Burman P„ Landolph J.R. Enhanced expression and state of the c-myc oncogene in chemically and X-ray-transformed C3H/10T1/2 CI 8 mouse embryo fibroblasts. Cancer Res. 1987; 47:3643-3649.
Bishop J.M. The molecular genetics of cancer. Science 1987; 235:305-311.
Boccara M., Souyri M., Magarian C, Stavnezer E., Fleissner E. Evidence for a new form of retroviral env transcript in leukemic and normal mouse lymphoid cells. J. Virol. 1983;48:102-109.
Borek C. The induction and control of radiogenic transformation in vitro: cellular and molecular mechanisms. Pharmac. Ther. 1985; 27:99-142.
Borek C. Radiation and chemically-induced transformation: free radicals, antioxidants and cancer. Br. J. Cancer 1987a; 55:74-86.
Borek C.The induction J radiogenic transformation in vitro: cellular and molecular mechanisms. Intern. Encyclopedia of Pharmacology and Therapeutics. In: Grunberger D., Goff S.P., eds. New York: Pergamon Press, 1987b: 151-196.
43 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
Borek C. Oncogenes, hormones, and free-radical processes in malignant transformation in vitro. Annals of the New York Academy of Sciences 1988; 95-102.
Borek C, Ong A., Mason H. Distinctive transforming genes in X-ray-transformed mammalian cells. Proc. Natl. Acad. Sci. USA 1987a; 84:794-798.
Borek C, Ong A., Mason H. In vitro transformation of human cells by radiation. Toxicol. Ind. Health 1987b; 3:397-404.
Borek C, Ong A., Mason H. Detecting dominant transforming genes in cells transformed in utero. Toxicol. Ind. Health 1988; 4: 541-545.
Borek C, Sachs L. The number of cell generations required to fix the transformed state in X-ray-induced transformation. Proc. Natl. Acad. Sci. USA 1968; 59:83-85.
Bryant P.E. Enzymatic restriction of mammalian cell DNA using PvuII and Bam HI: evidence for the double-strand break origin of chromosomal aberrations. Int. J. Radial Biol. 1984; 46: 57-65.
Chadwick K.H., Leenhouts H.P. The Molecular Theory of Radiation Biology. Heidelberg: Springer Verlag, 1981.
Chan G.L., Little J.B. Neoplastic transformation in vitro. In: Upton A.C., Albert R.E., Burns F.J., Shore R.E., eds. Radiation Carcinogenesis. New York: Elsevier, 1986:107-136.
Cohen J.J., Duke R.C. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei to cell death. J. Immunol. 1984; 132:38-42.
Cohen J J., Duke R.C., Chervenak R., Sellins K.S., Olson L.K. DNA fragmentation in targets of CTL: an example of programmed cell death in the immune system. Adv. Exp. Med. Biol. 1985;184:493-506.
Coni P., Pichiri-Coni G., Ledda-Columbano G.M., Rao P.M., Rajalakshmi S., Sarma D.S.R., Columbano A. Liver hyperplasia is not necessarily associated with increased expression of c-fos and c-myc mRNA. Carcinogenesis 1990; 11:835-839.
Cooper G.M., Lane M.A. Cellular transforming genes and oncogenesis. Biochem. Biophys. Acta 1984; 738:9-20.
Cooper G.M., Neiman P.E. Transforming genes of neoplasms induced by avian lymphoid leukosis viruses. Nature 1980; 287:656-659.
44 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
Cornforth M.N., Meyne J„ Littlefield L.G„ Bailey S.M., Moyzis R.K. Telomere staining of human chromosomes and the mechanism of radiation-induced dicentric formation. Rad. Res. 1989; 120:205-212.
D'Ambrosio S.M., Setlow R JB. Enhancement of postreplication repair in Chinese hamster cells. Proc. Natl. Acad. Sci. USA 1976; 73:2396-2400.
D'Ambrosk) E^ Waitzkin S.D^ Witney VJl., Salemaw A„ Furano A.V. Structure of the highly repeated, long interspersed DNA family (LINE or LIRn) of the rat. Mol. Cell. Biol. 1986;6:411-424.
Deragon J.-M„ Sinnett D., Labuda D. Reverse transcriptase activity from human embryonal carcinoma cells NTera2Dl. EMBO1.1990; 9: 3363-3368.
Diamond L.E., Guerrero I., Peflker A. Concominant K- and N-ras gene point mutations in clonal murine lymphoma. Mol. Cell. Biol. 1988; 8:2233-2236.
Druker BJ., Mamon HJ., Roberts TJVf. Oncogenes, growth factors, and signal transduction. New England J. Med. 1989; 321:1388-1391.
Dudley J.P. Discrete high molecular weight RNA transcribed from the long interspersed repetitive element LIMd. Nucleic Acids Res. 1987; 1 S: 2581-2S92.
Duke R.C., Chervenak R., Cohen JJ. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc. Natl. Acad. Sci. USA 1983; 80:6361-6365.
Duptan J.F., ed. Radiation-induced Leukemogenesis and Related Viruses. Amsterdam: Inserm Symposium 4,1977.
Economou-Pachnis A., Lohse M.A„ Furano A.V„ Tsichlb P.N. Insertion of long interspersed repeated elements at the Igh (immunoglobulin heavy chain) and Mlvi-2 (Moloney leukemia virus integration 2) loci in rats. Proc. Natl. Acad. Sci. USA 1985; 82: 2857-2861.
Edgell M.H., Hardies S.C., Loeb D.D., Shenee W.R., Padgett R.W., Burton F.H„ Comer M.B., Casavant N.C., Funk F.D., Hutchison III C.A. The LI family in mice. In: Stamatoyannopolos G., Nienhuis A.W., eds. Developmental Control of Globin Gene Expression. New York: Alan R. Liss, Inc. 1987:107-129.
Fanning T,, Singer M. The LINE-1 DNA sequences in four mammalian orders predict proteins that conserve homologies to retrovirus proteins. Nucleic Acids Res. 1987; 15: 2251-2260.
45 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
Farber E. Cellular biochemistry of the stepwise development of cancer with chemicals: G.H.A. Gowes Memorial Lecture. Cancer Res. 1984; 44:5463-5474.
Franklin W.A., Lo K.M., Hasettine WA. Alkaline lability of fluorescent photoproducts produced in ultraviolet light-irradiated DNA. J. Biol. Chem. 1982; 257:13535-13543.
Friend S.H., Bernards R., Rogelj S., Weinberg R.A., Rapaport JLM„ Albert DJH., Dryja T.P. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986; 323:643-646.
Fry R J.M. Experimental radiation carcinogenesis: What have we learned? Radiat. Res. 1981;87:224-239.
Furano A.V., Robb S.M., Robb F.T. The structure of the regulatory region of the rat LI (LIRn, long interspersed repeated) DNA family of transposable elements. Nucleic Acids Res. 1988; 16:9215-9231.
Furuno-Fukushi I., Matsudaira H. Mutation induction by different dose rates of y-rays in radiation-sensitive mutants of mouse leukemia cells. Rad. Res. 1989; 120:370-374.
Garte S J., Sawey M J., Burns F J. Oncogenes activated in radiation-induced rat skin
tumors. In: Radiation Carcinogenesis and DNA Alterations. Burns FJ.t Upton A.C., Silini G„ eds. Plenum Press, 1986:389-399.
Goodhead D.T. The initial physical damage produced by ionizing radiation. Int. J. Radiat. Biol. 1989; 56:623-634.
Guerrero I., Pellicer A. Mutational activation of oncogenes in animal model systems of carcinogenesis. Mutation Res. 1987; 185:293-308.
Guerrero I., Villasante A., Corces V,, Pellicer A. Activation of a c-K-ras oncogene by somatic mutation in mouse lymphomas induced by gamma radiation. Science 1984; 225: 1159-1162.
Hall E J. Radiobiology for the Radiologist. New York: J.B. Lippincott Company, 1988.
Han A., Elkind MAI. Transformation of mouse C3H/10T1/2 cells by single and fractionated doses of X-rays and fission-spectrum neutrons. Cancer Res. 1979; 39: 123-130.
Hattori M., Hidaka S., Sakaki Y. Sequence analysis of a Kpnl family member near the 3' end of human B-globin gene. Nucleic Acids Res. 1985; 13:7813-7827.
46 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
Hattori M.t Kuhara S„ Takenaka 0., Sakaki Y. LI family of repetitive DNA sequences in primates may be derived from a sequence encoding a reverse transcriptase-related protein. Nature 1986; 321:625-628.
Hfll C.K., Carnes B.A., Han A., Elkind M.M. Neoplastic transformation is enhanced by multiple low doses of fission-spectrum neutrons. Radiation Res. 1985; 102:404-410.
Hill C.K., Han A., Elkind M.M. Promotion, dose rate, and repair processes in radiation-induced neoplastic transformation. Radiation Res. 1987; 109:347-351.
Hollis D.E., Schetbner A. Ultrastructural changes in epidermal Langerhans cells and melanocytes in response to ultraviolet irradiation, in Australians of Aboriginal and Celtic descent. Br. J. Dermatology 1988; 119:21-31.
Houck CM., Rinehart F.P„ Schmid C.W. A ubiquitous family of repeated DNA sequences in the human genome. J. Mol. Biol. 1979; 132:289-306.
IAEA (International Atomic Energy Agency). Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assesment. Technical Reports Series No. 20, IAEA, Vienna, 1986.
ICRP 26. Recommendations of the International Commission on Radiological Protection. ICRP Publication 26. Annals of the ICRP, Pergamon Press, Oxford, 1977.
ICRP 58. RBE for Deterministic Effects. Publication 58. Annals of the ICRP, Pergamon Press, Oxford, 1990.
Janowski M., Cox R., Strauss P.G. The molecular biology of radiation-induced carcinogenesis: thymic lymphoma, myeloid leukaemia and osteosarcoma. Int. J. Radiat. Biology 1990; 57:677-691.
Jenkins JR., Sturzbecher H.-VV. The p53 oncogene. In: Reddy E.P., Skalka A.M., Curran T., eds. The Oncogene Handbook. Amsterdam: Elsevier Science Publisher B.V., 1988: 403-423.
Joshi G.P., Nelson W J., Revell S.H., Shaw C.A. X-ray-induced chromosome damage in live mammalian cells, and improved measurements of its effect on their colony-forming ability. Int. J. Radiat. Biol. 1982; 41:161-181.
Kakunaga T. Requirement for cell replication in the fixation and expression of the transformed state in mouse cells treated with 4-nitroquinoline-l -oxide. Int. J. Cancer 1974; 14:736-742.
47 FINNISH CENTRE TOR RADIATION AND NUCLEAR SAFETY STUK-A100
Kamada N,, Uchino H. Chronologic sequence in appearance of clinical and laboratory findings characteristic of chronic myelocytic leukemia. Blood 1978; 51:843-850.
Kato H., Shimizu V., Schull WJ. Radiation-induced cancer risks among A-bomb survivors: the new dose system. In: Baverstock K.F., StatherJ.W., eds. Low Dose Radiation: Biological Bases of Risk Assessment. London: Taylor & Francis, 1989:19-27.
Katzir N„ Rechavi G„ Cohen J.B., Unger T., Simoni F., Segal S„ Cohen D., Givol D. "Retroposon" insertion into the cellular oncogene c-myc in canine transmissible venereal tumor. Proc. Natl. Acad. Sci USA 1987; 82:1054-1058.
Kazazian H.H., Wong C, Youssoufian H., Scott A.F., PhiUips D.G., Antooarakis S.E. Haemophilia A resulting from de novo insertion of LI sequences represents a novel mechanism for mutation in man. Nature 1988; 332:164-166.
Kelly G., Kerkof P.R., Brooks A.L., Haley PJ. Oncogene activation in radiation-induced lung tumours. In: Raverstock K.F., Slather J.W.,eds. Low Dose Radiation: Biological Bases of Risk Assessment. London: Taylor & Francis, 1989:286-295.
Kennedy A.R. Promotion and other interactions between agents in the induction of transformation in vitro in fibroblasts. In: SlagaTJ.,ed. Mechanisms of Tumor Promotion. Boca Raton: CRC Press Inc. 1984:13-55.
Kennedy A.R. Evidence that the first step leading to carcinogen-induced malignant transformation is a high frequency, common event. Carcinogenesis 1985; 9:355-364.
Kennedy A.R., Cairns Jn Little J.B. Timing of steps in transformation of C3H 10T1/2 cells by X-irradiation. Nature 1984; 307:85-86.
Kennedy A.R., Little J.B. Investigation of the mechanism for enhancement of radiation in vitro by 12-0-tetradecanoylphorbol-13-acetate. Carcinogenesis 1980; 1:1039-1047.
Kennedy A.R., Little J.B, Evidence that a second event in X-ray-induced oncogenic transformation in vitro occurs during cellular proliferation. Radiat.Res. 1984;99:228-248.
Kerr J.F.R., Wyllie A.H., Currie A.R. Apoptosis: a basic biological phenomenon with wide-ranging implication in tissue kinetics. Br. J. Cancer 1972; 26:239-257.
Kerr J.F.R., Searle J., Harmon B.V., Bishop CJ. Apoptosis. In: Potten C.f ed. Perspectives on Mammalian Cell Death. Oxford: Oxford University Press, 1987; 93-128.
48 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
Kirschmeier P., Gattoni-Celli S., Dina P., Weinstein I.B. Carcinogen- and radiation-transformed C3H 10T1/2 cells contain RNAs homologous to the long terminal repeat sequence of a murine leukemia virus. Ptoc. Natl. Acad. Sci. USA 1982; 79: 2773-2777. de Klein A., Geurts van Kessel A., Grosveld G., Bartram C.R., Hagemeijer A., Bootsma D., Spurr N.K„ Heisterkamp N., Groffen J., Stephenson JJt. A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukaemia. Nature 1982; 300:765-767.
Klein G., Klein E. Oncogene activation and tumor progression. Carcinogenesis 1984; 5: 429-435.
Knudson A.G. Hereditary cancer, oncogenes, and antioncogenes. Cancer Res. 1985; 45: 1437-1443.
Kuff E.L„ Feenstra A., Lueders K., Smith L., Hawley R., Hozumi N., Shulman M. Intracistemal A-parucle genes as movable elements in the mouse genome. Proc. Natl. Acad. Sci. USA 1983; 50:1992-1996,1983.
Lakshmikumaran M.S., D'Ambrosio E., Laimins L.A., Lin D.T., Furano A.V. Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus. Mol. Cell Biol. 1985; 5:2197-2203.
Land HU Parada L.F., Weinberg R.A. Cellular oncogenes and multistep carcinogenesis. Science 1983a; 222:771-778.
Land H., Parada. L.F., Weinberg R.A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 1983b; 304; 596-602.
Lieberman M.W., Beach L.R., Palmiter R.D. Ultraviolet radiation-induced metallothionein-I gene activation is associated with extensive DNA methylation. Cell 1983; 35:207-214.
Little J.B. Radiation carcinogenesis in vitro: implications for mechanisms. In: Hiat H.H., Watson, and Winston, eds. The Origins of Human Cancer. Cold Spring Harbor Conferences on Cell Proliferation. New York: Cold Spring Harbor Laboratories, 1977,923-939.
Little J.B. Quantitative studies of radiation transformation with the A31-11 mouse BALB/3T3 cell line. Cancer Res. 1979; 39: 1474-1480.
Lloyd D.C., Edwards A.A. Chromosome aberrations in human lymphocytes: Effects of radiation quality, dose and dose rate. In: Ischihara T„ Sakaki M.S., eds. Radiation-induced Chromosome Damage in Man. New York: Liss, 1983:23-49.
49 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
Lloyd E.L., Gemmell M.A., Henning C.B-, Gemmel) D.S., Zabransky B J. Transformation of mammalian cells by alpha particles. Int. J. Radial. Biol. 1979; 36: 467-478.
Loeb D.D., Padgett R.W„ Hardies S.C., Shehee W.R., Comer MB., Edge» M.H., Hutchison III C.A. The sequence of a large LIMd element reveals a tandemly repeated 5' end and several features found in retrotransposons. Mol. Cell Biol. 1986; 6:168-182.
Lu L.-J/W., Liehr J.G., Sirbasku D.A., Randerath E„ Randerath K. Hypomethylation of DNA in estrogen-induced and »dependent hamster kidney tumors. Carcinogenesis 1988; 9:925-929.
Maniatis T., Fritsch E.F., Sambrook J., eds. Molecular Cloning, A Laboratory Manual. New York: Cold Spring Harbor Laboratory, 1982.
Marchal C J., Rigby P.W. Viral and cellular genes involved in oncogenesis. Cancer Surv. 1984;3:183-214.
Miller R., Hall E J. X-ray dose fractionation and oncogenic transformations in cultured mouse embryo cells. Nature 1978; 272:58-60.
Mizuki K„ Nose K., Okamoto H., Tsuchida N., Hayashi K. Amplification of c-Ki-ras gene and aberrant expression of c-myc in WI-38 cells transformed in vitro by y-irradiation. Biochem. Biophys. Res. Comm. 1985; 128:1037-1043.
Moore J.V. Death of cells and necrosis of tumours. In: Potien C„ ed. Perspectives in Mammalian Cell Death. Oxford: Oxford Science Publication, 1987:295-325.
Morse B., Rotherg P.G., South VJ., Spandorfer J.M„ Astrin S.M. Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma. Nature 1988; 333: 87-90.
Moustacchi E., Ehmann U.K., Friedberg E.C. Defective recovery of semi-conservative DNA synthesis in Xeroderma pigmentosum cells following split-dose ultraviolet irradiation. Mutat. Res. 1979; 62:159-171.
Natarajan A.T., Obe GM Van Zeeland A.A., Palitti F., Meijers M,, Verdegaal-Immerzeel E.A.M. Molecular mechanisms involved in the production of chromosomal aberrations, II, Utilization of Neurospora endonuclease for die study of aberration production by X-rays in Gi and G2 stages of the cell cycle. Mutat Res. 1980; 69:293-305.
NCRP Report 106. Limit for exposure to "hot particles" on the skin. National Council on Radiation Protection and Measurements, 1990.
50 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
Nur I., Pascale E., Furano A.V. The left end of ratL I (LlRn, long interspersed repeated) DNA which is a CpG island can function as a promoter. Nucleic Acids Res. 1988; 16: 9233-9251.
Orr-Weaver T.L., Spradling A.C. Drosophila chorion gene amplification requires an upstream region regulating sl8 transcription. Mol. Cell Biol. 1986; 6:4624-4633.
Olson R.L., Everett M.A. Epidermal apoptosis: Cell deletion by phagocytosis. J. Cutaneous Pathology 1975; 2:53-57.
Padgett R.W., Hutchison III C.A., Edgell M.H. The F-type 5' motif of mouse LI -elements: a major class of LI termini similar to the A-type in organization but unrelated in sequence. Nucleic Acids Res. 1988; 16:739-749.
Painter R.B. The role of DNA damage and repair in cell killing induced by ionizing radiation. In: Meyn R.E. and Withers H.R., eds. Radiation Biology in Cancer Research. New York: Raven Press, 1979:59-68.
Peak M J., Peak J.G. Solar-ultraviolet-induced damage to DNA. Photodermatology 1989; 6: 1-15.
Pear W.S„ Nelson S.F., Axelson H„ Wahlström &, Bazin H„ Klein G., Sumegi J. Aberrant class switching juxtaposes c-myc with a middle repetitive element (LINE), and an IgH intron in two spontaneously arising rat immunocytomas. Oncogene 1988; 2: 499-507.
Preston D.L., Kato H., Kopecky K J., Fujita S. Studies of the mortality of A-bomb survivors.Rad.Res. 1987; 111: 151-178.
Radford I.R., Hodgson G.S., Matthews J.P. Critical DNA target size model of ionizing radiation-induced mammalian cell death. Int. J. Radiat Biol. 1988; 54:63-79.
Rajewsky B., Franke Th., Groos E„ Heyder J„ Kaul A., Lippert W„ Rajewsky M.F. "Heisse teilchen". Untersuchung radiaktiver Partikel aus dem Jahre 1961. Atompraxiä 1962; 7:1-24.
Reznikoff C.A., Bertram J.S., Brankow D.W., Heidelberger C. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res. 1973a; 33:3239-3249.
Reznikoff C.A., Brankow D.W., Heidelberger C. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 1973b; 33: 3231-3238.
51 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-A100
Rogers J.H. The origin and evolution of retroposons.Int . Rev. Cytol. 1985; 93:187-279.
Rogers J. Relationship of Blym genes to repeated sequences. Nature 1986; 320:579.
Roots R^ Kraft G., Gosshalk E. The formation of radiation-induced DNA breaks: The ratio of the double-strand breaks to single-strand breaks. Int. J. Radiation Oncology Biol. Phys. 1985; 11:259-265.
Sawey M J., Hood A.T., Burns F J., Garte S J. Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation. Mol. Cell. Biol. 1987; 7: 932-935.
Sawey MJ., Kennedy A.R. Activation of oncogenes in radiation-induced malignant transformation. In: Baverstock K.F., Stather J.W., eds. Low Dose Radiation: Biological Bases of Risk Assessment London: Taylor & Francis, 1989:433-438.
Schmidt J., Erfle V., Miiller W.A. Activation of endogenous C-type retroviral genomes by internal alpha-irradition of mice with 324radium. Radiat. Environ. Biophys. 1985; 24: 17-25.
Schön A., Michiels L., Janowski M., Merregaert J., Erfle V. Expression of protooncogenes in murine osteosarcomas. Int. J. Cancer 1986; 38:67-74.
Searle J., Kerr J.F.R., Bishop C J. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol. Ann. 1982; 17:229-259.
Servomaa K., Rytömaa T. Observations on DNA damage caused by UV and ionizing radiation. 17th Meeting of the European Study Group for Cell Proliferation, Helsinki, 1990: 93.
Shay H., Gruenstein M., Harris C. Experimental leukemias in the rat. Their development and transfer and some applications to the human problem. Acta Haemat. 1955; 14:337-353.
Shi Y., Sahai B.M., Green D.R. Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes. Nature 1989; 339:625-626.
Singer M.F, Highly repeated sequences in mammalian genomes. Int. Review Cytol. 1982; 76:67-112.
Singer M.F. SINE and LINE nomenclature. TIG 1990; 6:204.
Singer M.F., Thayer R.E., Grimaldi G., Lerman M.I., Fanning T.G. Homology between the Kpnl primate and Bam HI (M1F-1) rodent families of long interspersed repeated sequences. Nucleic Acids Res, 1983; 11:5739-5745.
52 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY
Sinkko K, Aaltonen H. Calculation of the true coincidence summing correction for different sample geometries in gamma-ray spectroscopy. Report STUK-B-VALO 40, Finnish Centre for Radiation and Nuclear Safety, 1985 (in Finnish).
Skowronski JL, Singer M.F. Expression of a cytoplasmic LINE-1 transcript is regulated in a human teratocarcinoma cell line. Ftoc. Natl. Acad. Sei I985; 82:6050-6054.
Smith C.A„ Williams G.T., Kingston R„ Jenkinson EJ., Owen JJ.T. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 1989; 337:181-184.
Sorrentino V., Drozdoff V., Zeitz L., Fleissner E. Increased radiation-induced transformation in C3H/10T1/2 cells after transfer of an exogenous c-myc gene. Ptoc. Natl. Acad. Sci. USA 1987; 84:4131-4134.
Stanbridge E J., Nowell P.C. Origins of human cancer revised. Cell 1990; 63:867-874.
Sturm S.A., Strauss P.G., Adolph S., Hameister H., Erfle V. Amplification and rearrangement of c-myc in radiation-induced murine osteosarcomas. Cancer Res. 1990; 50:4146-4153.
Suzuki K., Suzuki F., Watanabe M., Nikaido O. Multistep nature of X-ray-induced neoplastic transformation in Golden hamster embryo cells: Expression of transformed phenotypes and stepwise changes in karyotypes. Cancer Res. 1989; 49:2134-2140.
Swergold G.D. Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol. Cell. Biol. 1990; 10:6718-6729,
Terasima T., Yasukawa M., Kimura M. Radiation-induced transformation of 10T1/2 mouse cells in the plateau phase: Post-irradiation changes and serum dependence. Gann 1981;72:762-768.
Terzaghi MM Little J.B. Oncogenic transformation in vitro after split-dose x-irradiation. Int. J. Radiat Biology 1976; 29:583-587.
TrgovcevilZ., Lers N., Brcil-KostilK., Salaj-Smi Truneh A., Albert F., Golstein P., Schmitt-Verhutst A.-M. Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature 1985:313:318-320. 53 FINNISH CENTRE FOR RADIATION AND NUCLEAR SAFETY STUK-AIOO Upton A.C. Biological aspects of radiation carcinogenesis. In: Boice J.D., Fraumeni J.F., eds. Radiation Carcinogenesis: Epidemiology and Biological Significance. New York: Raven Press 1984:9-19. Upton A.C. Biological basis of radiological protection and its application to risk assessment. Br. J. Radiol, 1987; 60:1-16. van der Rauwelaert, Maisin J.R., Merregaert J. Provirus integration and myc amplification in ^Sr-induced osteosarcomas of CF1 mice. Oncogene 1988; 2:215-222. Varmus H. A historical overview of oncogenes. In: Weinberg R.A., ed. Oncogenes and the molecular origins of cancer. New York: Cold Spring Laboratory Press, 1989:3-44. Voliva C.F., Jahn C.L., Comer M.B., Hutchison m C.A., Edgell M.H. The LlMd long interspersed repeat family in the mouse: almost all examples are truncated at one end. Nucleic Acids Res. 1983; 11:8847-8859. Voliva C.F., Martin S.L., Hutchison HI C.A., Edged M.H. Dispersal process associated with the LI family oi interspersed repetitive DNA sequences. J. Mol. Biol. 1984; 178: 795-813. Waldren C, Correll L., Sognier M.A., Puck TX Measurement of low levels of X-ray mutagenesis in relation to human disease. Proc. Natl. Acad. Sci. USA 1986; 83:4839-4843. Ward J.F. DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation and reparability. Progress Nucleic Acids Mol. Biol. 1988; 35: 95-125. Ward J.F. The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review. Int. J. Radiat. Biol. 1990; 57:1141-1150. Warnock R. V., Bray L.G., Cooper T.L., Goldin E.M., Knapp PJ., Lewia M.M„ Rigby W.F. A health physics program for operation with failed fuel. Radiat. Prot. Manag. 1987; 4:21. Weber J„ McClure M. Oncogenes and cancer. Br. Med. J. 1987; 294:1246-1248. Weedon D., Searle J., Kerr J.F.R. Apoptosis: its nature and implications for dermatopathology. American J. Dermatopathlogy 1979; 1:133-144. Weinberg R.A. The action of oncogenes in the cytoplasm and nucleus. Science 1985; 230: 770-776. Weinberg R.A. Finding the anti-oncogene. Scientific American 1988; September: 34-41, 54 FINNISH CENTRE FOR RADIATION STUK-A100 AND NUCLEAR SAFETY Weinberg R.A. Oncogenes, anttoncogenes, and the molecular bases of multistep carcinogenesis. Cancer Res. 1989; 49: 3713-3721. Weiner A.M., Deiniger P.L., Efstratiadis A. Nonviral retroposons: genes, pseudogenes, and transposable elements generated by the reverse flow of genetic information. Ann. Rev. Biochem. 1986;55:631-661. Wikjram G.F., Kidd R.L„ Krawczyk W.S., Cole PX. Sunburn effect on keratinosomes: A reportwit h special note on ultraviolet-induced dyskeratosis. Arch. Derm. 1970; 101: 505-519. Wilson V.L., Smith R. A„ Ma S., Cutter R.G. Genomic 5-methyldeoxycytidine decreased with age. J. Biol. Chem. 1987; 262:9948-9951. Wright N.A. Cellular pathology as endpoints in cell transformation assays. In: Chadwick K.H., Seymour C, Bamhart B„ eds. Cell Transformation and Radiation-induced Cancer. Bristol: Adam Hilger 1989:11-16. Wyllie A.H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980; 284:555-556. Wyllie A.H. Apoptosis: cell death in tissue regulation. J. Pathol. 1987; 153:313-316. Wyllie A.H., Kerr J.F.R., Currie A.R. Cell death: the significance of apoptosis. Int. Rev. Cytol. 1980; 68:251-306. Wyllie A.H., Morris R.G., Smith A.L., Dunlop D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathology 1984; 142:67-77. Yamada T., Ohyama H. Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis). Int. J. Radiat. Biol. 1988; 53:65-75. 55 I RYTÖMAA T.f SERVOMAA K., TOIVONEN, H. Chernobyl reactor accident and radioactive particles found in Finland. Duodecim 1986; 102: 1190-1196 (in Finnish). RYTÖMAA T., SERVOMAA K., TOIVONEN, H. Chernobyl reactor accident and radioactive particles found in Finland. Duodecim 1986; 102: 1190-1196 (in Finnish). DUODECIM 102: 1190—1196, 1986 TSERNOBYLIN YDINVOIMALATURMASSA SYNTYNEET KUUMAT HIUKKASET: MAHDOLLINEN VAARA TERVEYDELLE SUOMESSA Tapio Rytömaa, Kristina Servomaa ja Harri Toivonen Tsernobylin y di n voimalat urman Suomessa teilyn terveydelle aiheuttama haitta yleensä aiheuttamaa radioaktiivista laskeumaa on seu yritetään arvioida, on sopimuksenomainen rattu laajan ja yksityiskohtaisen mittausjär laskutoimitus. Terveyshaitan arvioiminen an- jestelmän avulla (STUK-B-Valo 1986 a, b). nosekvivalentista on epävarmaa nykyisen bio Mitatuista säteilynopeuksista ja ympäristö- lääketieteellisen ja epidemiologisen tiedon näytteiden sisältämien radionuklidien pitoi avulla ja lisäksi Tsernobylin ydinvoimalatur- suuksista voidaan likimääräisesti arvioida se maan liittyy ainakin yksi erityispiirre, joka te säteilyannos, jonka suomalainen on laskeu kee keskimääräisiin annoksiin perustuvan ar masta jo saanut ja tulevaisuudessa vielä kes vioinnin entistäkin vaikeammaksi ja epämää- kimäärin saa. Nykyisen arvion mukaan kes räisemmäksi. Esitämme tässä artikkelissa kimääräinen kokonaisannos vastaisi laskeu alustavat havainnot uuden, nyt ensimmäistä ma-alueella suunnilleen yhdessä vuodessa kertaa todetun ilmiön luonteesta ja sen mah luonnonsäteilystä saatua annosta. Englannissa dollisesti aiheuttamasta terveyshaitasta. on arvioitu, että siellä laskeuman aiheuttama lisäannos olisi ensimmäisenä vuotena vain Kuumien hiukkasten etsintä noin 4 % vuotuisesta luonnonsäteilyannokses- ta ja myöhempinä vuosina huomattavasti vä Tsernobylin ydinvoimalaturmassa reak- hemmän (Fry ym. 1986). torisydämen lämpötila nousi paikallisesti Säteilyannosten muuttaminen efektiivisik 2 500°C:seen ja ehkä vieläkin korkeammak si annosekvivalenteiksi (ICRP 1977), joista sä si. Monet huonosti höyrystyvät nuklidit, ku- Tsernobylin ydinvoimalaturmassa syntyneet kuumat hiukkaset nti ten zirkoniumin, ruteniumin ja ceriumin iso dak X-OMAT AR). Järjestelmä on sama, jo toopit, sulivat ja vapautuivat reaktorin sydä ta käytetään yleisesti esimerkiksi geeniteknii mestä ja muodostivat jäähtyessään erikokoi kassa tutkittaessa nukleiinihappoja 3ZP:lla sia aerosolihiukkasia. Korkealle ilmaan jou merkittyjen koettimien avulla. Valotusaika tuneet aerosolit kulkeutuivat sitten ilmavirto vaihteli yhdestä minuutista viiteen vuorokau jen mukana pitkien matkojen päähän, ja oli teen kiinnostavan kuuman hiukkasen aktiivi mahdollista, että tällaisia hiukkasia löytyisi suuden mukaan. Filmit kehitettiin sen jälkeen Suomesta. Viitteitä erilaisten aerosolihiukkas rutiinimenetelmällä (Sakura QX 60). ten olemassaolosta saatiin onnettomuuden jäl keen jo varhaisessa vaiheessa Valtion teknil Hiukkasten koostumus ja määrä lisen tutkimuskeskuksen, Ilmatieteen laitok sen ja Säteilyturvakeskuksen tekemistä ilma Varsin tyypillinen löydös leikatusta nurmi näytteiden mittauksista (Kauppinen ym. kosta, johon oli kertynyt sadevettä mm. ra 1986); ruotsalaisten Studsvtkissä tekemät sa kennusten katoilta, on esitetty kuvassa 1. Täs mankaltaiset havainnot on jo ehditty julkais- sä pitkään valotetussa (5 vrk) näytteessä ol takin (Devell ym. 1986). leen tuoreen ruohon ääriviivat näkyvät selvästi Me olemme etsineet laskeuman sisältämiä ruohon sisältämien cesiumin isotooppien 134 »kuumia hiukkasia», mikroskooppisen pieniä ja 137 vuoksi; '"jodin määrä näytteessä oli metallikappaleita, suoraan luonnosta. Tällä autoradiografiavaiheessa jo hyvin vähäinen. tavalla voidaan saada käsitys hiukkasten ra- dionuklidikoostumuksesta, aktiivisuudesta, kokovaihtelusta, esiintymistaajuudesta ja myös hiukkasiin mahdollisesti liittyvästä ter veyshaitasta. Hiukkasia etsittiin alustavasti Jm K ">**_,' > T pintakontaminaatiomittarilla (Wallac RD-11), (Jm '••-' f ***M<•- jolla paikallistettiin sellaisia pieniä maa-alueita AU . / W } ja kasvustoja, joissa säteilynopeus oli selväs ti suurempi kuin ympäristössä. Mm. Uuden Varsinaiset kuumat hiukkaset erottuvat ku tiivisuudet ovat aivan ilmeisesti johtuneet vassa pieninä mustina pisteinä ja isompina näytteen epäpuhtauksista. Toistaiseksi suu täplinä. Täplän pinta-ala on verrannollinen rimman eristämämme kuuman hiukkasen ko- hiukkasen radioaktiivisuuteen. Kaikkien ku konaisaktiivisuus huhtikuun lopussa oli noin vassa näkyvien hiukkasten todellinen koko 1 000 Bq. (radionuklidipitoisuudesta laskettuna) on pie Kuvassa 2 on esitetty sammalnäyte ja siitä nempi kuin 1 /im. tehty autoradiogrammi. Näyte on kerätty Suurimmat löydetyt kuumat hiukkaset, joi aukealta paikalta, johon sadevettä ei ole vir den aktiivisuus oli kymmenkertainen tai enem rannut muualta, ja näyte edustaa siten tälle män suurimpaan kuvassa I näkyvään hiukka alueelle tullutta suoraa laskeumaa. Sammal on seen verrattuna, on pyritty eristämään gam- kuitenkin ilmeisesti poikkeuksellisen hyvin es maspektrometristä analyysiä varten. Eristämi tänyt kuumien hiukkasten huuhtoutumisen sa nen tehtiin yksinkertaisesti jakamalla hiukka teen mukana maaperään, ja hiukkasten luku sen sisältävä näytealue toistuvasti yhä pienem määrä maa-alueen pinta-alayksikköä kohti on piin osiin ja paikantamalla voimakkaasti ra suuri. Tarkkaa arviota hiukkasten esiintymis- dioaktiivinen mikrolähde autoradiografisesti. taajuudesta tällä alueella on mahdotonta teh Aktiivisimpien hiukkasten toteaminen on dä, mutta mm. näytteen sammalmäärästä mahdollista alle minuutin valotusajalla, joten päätellen suhteellisen helposti todettavia hiuk näiden hiukkasten eristäminen on varsin help kasia on enemmän kuin yksi neliösenttimet poa. rillä. Alustavien gammaspektrometristen mit tausten mukainen kuuman hiukkasen tyypil Mahdollinen terveyshaitta linen radionuklidikoostumus on esitetty tau lukossa 1. Yksittäisten hiukkasten kokonais- Mikä sitten on tällaisten hiukkasten poten aktiivisuudet vaihtelevat kuitenkin suuresti tiaalinen merkitys mahdollisten terveyshaitto hiukkasten koon mukaan; esimerkiksi kym jen kannalta? Kuvissa 1 ja 2 näkyviä hiukka menkertainen ero hiukkasten halkaisijassa sia kukaan ei varmaan mielellään ottaisi eli merkitsee laskennallisesti tuhatkertaista eroa mistöönsä eikä kaiketi soisi lehmänkään nii niiden aktiivisuudessa. tä syövän. Ruoansulatuskanavaan joutues Eristetyissä hiukkasnäytteissä ei ole yleen saan taulukossa 1 esitetyt radionuklidit sisäl sä todettu merkittäviä määriä cesiumin tai jo tävä metallipallo erittyisi kuitenkin hyvin to din isotooppeja. Näiden nuklidien pienet ak- dennäköisesti sellaisenaan ulos. Zirkonium, niobium, ruteniurn ja cerium ovat maa-alkali- metalleja, joiden stabiileja isotooppeja esiin Taulukko I. Eristetyn kuuman hiukkasen radio tyy ihmiselimistössä pieniä määriä (muutamia nuklidikoostumus. Suurimpien hiukkasten kokonaisak- kymmeniä tai satoja milligrammoja) tai niitä tiivisuus voi olla yli satakertainen tähän hiukkaseen ver ei esiinny lainkaan. Näiden aineiden imeyty rattuna (ks. myös Devell ym. 1986). minen suolistosta elimistöön — sikäli kun asia Nuklidi Puoliintumis Aktii tunnetaan (ICRP 1975) — on hyvin vähäistä aika visuus erilaisina suoloinakin. Kiinteästä ja veteen liu (vrk) (Bq) kenemattomasta metallipallosta absorptiota ei "Zirkonium 64 26 luultavasti voi tapahtua lainkaan. Toisaalta on ""Ruteniurn 39 43 kuitenkin mahdollista, että tällainen pieni me- ,0*Rutenium 368 9.3 l4lC'erium 32 22 tallinsiru (ks. myös Devell ym. 1986) saattaa '"Cerium 284 18 jäädä pitkäksi aikaa kiinni johonkin ruoan- Tshernobylin ydinvoimalaturmassa syntyneet kuumat hiukkaset 11» Kuva 2. Sammalnäyte (A), josta tehty autoradiogrammi (B) osoittaa, että suorassakin laskeumassa on paikoin paljon kuumia hiukkasia. Valotusaika 2 vrk, kuvan pienennys 1:3. sulatuskanavan limakalvon osaan, esimerkiksi nen — ja ilmeisesti säteilymittarin toiminta villusten tyveen. Mahdollista on myös se, että häiriön laukaissut — säteilypulssi saattoi joh tuuli tai jokin muu tekijä nostaa irrallisen tai tua tuulen kuljettamasta kuumasta hiukka pölyyn kiinnittyneen hiukkasen maasta il sesta. maan, jolloin se saattaa joutua hengitysteihin Entä sitten, jos kuuma hiukkanen kiinnit ja aerosolin koon mukaan kiinnittyä siellä eri tyy hengitysteiden tai ruoansulatuskanavan li paikkoihin. Ohimennen todettakoon, että makalvoon? Kuva 3 antaa tästä tilanteesta Kotkassa 9. 6. 1986 mitattu hyvin lyhytkestoi melko konkreettisen ja mittasuhteiltaan aivan &! Kuva 3. Suhteellisen aktiivisen (noin 1 kBq) kuuman hiukkasen autoradiogrammi! 1,10, 100 ja 1 000 minuutin valotusaikojen jälkeen (ylempi kuvasarja). Valottuneen alueen laajuus antaa todellisen, mittasuhteiltaan oikean ku van säteilyenergian absorboitumisesta kuumaa hiukkasta ympäröivään kudokseen (kuva on alkuperäiskoossa). Noin 1 mm:n laajuista keskustaa lukuun ottamatta annos aiheutuu lähes kokonaan ,MRu:n ja IMCe:n tytärnuklidien ,nRrodiumin ("*Rh) ja '"proseodyymin ('"Pr) kovista beetahiukkasista. Alemmassa kuvasarjassa on esitetty pienem män kuuman hiukkasen auioradiogrammit, Tässä hiukkasessa heikkoenergiaisten beetasäteilijöiden osuus on ilmei sesti suhteellisen pieni. 1194 T. Rytömaa, K. Servomaa ja //. Toivonen todellisen käsityksen. Ylemmästä kuvasarjasta näkyy, miten valottuneen Filmin pinta-ala kas vaa valotusajan pidentyessä. Filmin mustumi nen kuvaa sitä annosta, jonka vastaavanko koinen kudospallo tästä säteilylähteestä sai si. Noin 17 tunnin (1 000 min) säteilytyksen jälkeen mustuman halkaisija on jo yli 2 cm, ja tämä osoittaa, että eristetty kuuma hiuk kanen sisältää runsaasti l06Ru:ta, ,44Ce:tä tai molempia (ruteniumin isotoopin 106 tytärnuk lidin "*rodiumin (Rh) keskimääräinen bee- taenergia on l 508 keV ja l44ceriumin tytär l44 nuklidin praseodyymin 1 222 keV. Muiden Pallon halkaisija 1 mm Pallon halkaisija 2 cm s taulukon 1 nuklidien beetasäteily absorboituu eli noin 5 x 10 solua eli noin 4 x 10* solua ja annosnopeus yli 100 ja annosnopeus n. 0.04 käytännössä täydellisesti hiukkasen lähietäi mGy/h (10 rad/h): solut mGy/h (4 m rad/h): solu saavat vuorokaudessa jen keskimääräinen an syyteen eli palloon, jonka halkaisija on tappavan sädeannok- nos vrk ssa 1 mGy (100 1 mm). Beetasäteilyn kantaman ulkopuolella mrad). kin (pallon maksimihalkaisija on noin 3 cm) Kuva 4. Annosnopeudet sellaisen kuuman hiukkasen elimistöön absorboituu hiukkasen lähettämäs ympärillä, jonka radionuklidikoosiumus on suunnilleen tä gammasäteilystä energiaa, mutta solujen sama kuin taulukossa 1. Hiukkasen kokonaisaktiivisuu- saama keskimääräinen annos jää kuitenkin deksi on tässä esimerkissä valittu I k Bq (aktiivisuus vas taa suunnilleen kuvan 3 ylärivissä esitetyn hiukkasen pieneksi. Alemmassa kuvasarjassa on esitet aktiivisuutta). ty toisen, vähemmän aktiivisen hiukkasen au- roradiogrammit. Tämä hiukkanen näyttää si sältävän suhteellisen vähän heikkoenergiais- ta beetasäieilyä lähettäviä radionuklideja, kos- nuklideja sisältävä pistemäinen säteilylähde ai ka yhden minuutin valotusajan jälkeen hiuk heuttaa erittäin selvän annosgradientin lähteen kasen autoradiografinen jälki on vielä hyvin ulkopuolelle. heikko. Jos hiukkanen sisältäisi paljon tällai Jakautumiskykyinen solukko kuolee lähes sia beetasäteilijöitä, noin 1 mmm ympyrän pi varmasti, jos sen saama annos suhteellisen no täisi mustua nopeasti. Tässä yhteydessä on peasti (muutamassa vuorokaudessa) ylittää 10 syytä huomata, että valikoimalla kuumat Gy:n määrän. Niinpä kaikki kuvassa 4 esite hiukkaset niiden filmillä aiheuttaman mustu tyn mustan alueen sisältämät noin viisisataa- man koon perusteella (yli ja alle 1 mm) hiuk tuhatta solua ilmeisesti kuolevat. Tällaisella kaset voidaan ilmeisesti jakaa paljon ja vähän solukuolemalla ei sellaisenaan ole kuitenkaan ruteniumia ja ceriumia sisältäviin. mitään ilmeistä merkitystä terveydelle, sillä ti Kuvassa 4 on kaavamaisesti esitetty sellai lanne vastaa patologisesti mikroskooppista sen kuuman hiukkasen ympäristön soluille an palovammaa. tamat annokset, jossa on suhteellisen paljon Kuolemapesäkkeen ulkopuolella on vyöhy lwRu:ta ja ,44Ce:tä, Tällainen hiukkanen on ke, jossa sofujen saama säteilyannos on suuri valittu alustavaksi esimerkiksi, koska tehty mutta ei tappava (kuvan 4 isossa pallossa kes jen autoradiogrammien ja gammaspektromet- kimäärin tosin vain 1 mGy/vrk). Annos on risten mittausten perusteella ainakin näitä mustan alueen lähellä kuitenkin hyvin suuri hiukkasia on Suomeen tulleessa laskeumassa ja pienenee sitten vähitellen, kun solun etäi melko paljon (ks. kuvat I ja 2) ja koska näitä syys radioaktiivisesta pesäkkeestä kasvaa. Tlemobylin ydinvoimalaturmassa syntyneet kuumat hiukkaset 1195 Pohdinta tämistä radionuklideista koko elimistölle las kettu keskimääräinen säteilyannos on har Jonkinlaisten kuumien hiukkasten mahdol haanjohtava suure, jolla ei ole mitään vasti linen olemassaolo radioaktiivisten aineiden ai netta todellisuudessa. heuttamassa kontaminaatiossa on tiedostettu Julkaistut kokeelliset tutkimukset, joilla jo monta vuotta sitten, ja aikoinaan asiasta kuumiin pisteisiin liittyvää mahdollista hait käytiin tieteellisessä ja puolitieteellisessä leh taa on pyritty vertaamaan kudokseen tasaises distössä varsin kiivasta väittelyä (NCRP ti jakautuneeseen säteilyannokseen, eivät tuo 1975). Tässä keskustelussa pohdittiin kuiten olennaista lisäselvyyttä nyt ilmenneeseen kin tilannetta, jossa kuuma hiukkanen olisi al- uuteen ongelmaan. Tästä syystä myös vertai fasäteilijä eikä tämä siten lainkaan vastaa ny lu »tavanomaisen» jodi- ja cesiumlaskeuman kytilannetta. Alfahiukkasen kantama kudok aiheuttamaan haittaan on vaikeaa, vaikka las sessa on hyvin lyhyt, ja hiukkanen pysähtyy keumien kokonaismäärät saattava! joillakin »yhtäkkiä». Lisäksi jo yksi ainoa osuma so alueilla olla lähes samanlaisia. Toisaalta on lun tumaan on melko todennäköisesti tappa kuitenkin selvää, että suurten (yli 1 /im) ja va (Munro 1970, NCRP 1975). Niinpä lähes mahdollisesti haitallisimpien kuumien hiuk kaikki solut, jotka ovat alfasäteilyä lähettä kasten lukumäärä laskeumassa on kaikkialla vän kuuman pisteen vaikutuspiirissä, saavat Suomessa pieni. »liian» suuren annoksen ja kuolevat. Sen si Osastopäällikkö Olli Paakkola Säteilytur jaan beeta- ja gammasäteilyä lähettävästä vakeskuksen valvontaosastolta saattoi tie mikroskooppisesta lähteestä miljoonat solut toomme, että samankaltaisia beeta-aktiivisia saavat säteilyannoksen, joka ei tapa soluja hiukkasia löydettiin myös ydinpommikokei- mutta joka on riittävän suuri aiheuttamaan den jälkeen 1950-ja 1960-lukujen vaihteessa. sattumanvaraisen pahanlaatuisen transfor Hiukkasten ominaisaktiivisuudet olivat kui maation. In vitro -olosuhteissa 1 Gy:n gam- tenkin paljon pienempiä kuin TSernobylin las masäteilyannos johtaa pahanlaatuisen kloo keumassa, sillä räjähdyksen yhteydessä syn nin kehittymiseen yhdestä solusta todennäköi tyneet aerosolit eivät sisältäneet pelkästään ra syydellä 10-'—10"' solutyypin ja olosuhtei dioaktiivisia aineita (ks. esim. Rajewsky ym. den mukaan (Kennedy ym. 1980, Hall ja Hei 1962). Näidenkään hiukkasten mahdollista 1985, Rytömaa ja Servomaa 1986). Huomat merkitystä terveydelle ei ole selvitetty tarkem takoon, että vaikka transformaatio tapahtui min. si näin suurella todennäköisyydellä myös in vivo, säteilyn aiheuttama kliininen syöpä ei kuitenkaan kehity yhtä helposti: jos se kehit tyisi, kaikkien ihmisten pitäisi saada syöpä jo Tiivistelmä luonnonsäteilystä, Tsernobylin ydinvoimalaturmassa Suomeen ti ote mitään ilmeistä syytä otaksua, että kulkeutui tavanomaisen laskeuman ohella nyt kuvattuihin kuumiin hiukkasiin ja niiden myös harvinaisia maa-alkalimetalleja sisältä mahdollisesti aiheuttamiin soluvaurioihin liit viä pieniä aerosoleja, joiden radioaktiivisuus tyisi välttämättä suurempi syöpäriski kuin sel oli hyvinkin suuri. Zirkoniumin, niobiumin, laiseen tilanteeseen, jossa absorboitunut sätei ruteniumin ja ceriumin isotoopeista koostu lyenergia on jakautunut tasaisesti kaikille so via kuumia hiukkasia voi suhteellisen helpos luille. Toisaalta taas on selvää, että kuumien ti löytää ja eristää luonnosta. Paikoin hiuk hiukkasten aiheuttama hyvin epätasainen an- kasten lukumäärä on jopa useita kymmeniä nosjakautuma merkitsee sitä, että niiden sisäl 100 cm!:n alueella. Beeta- ja gammasäteilyä i 1196 T. Rytömaa, K. Servomaa ja H. Toivonen lähettäviin kuumiin hiukkasiin mahdollisesti seksi epäselvä, ja tämä uusi asia vaatii yksi liittyvä potentiaalinen terveyshaitta on toistai tyiskohtaisia lisätutkimuksia. Kirjallisuutta Dcvell L, Tovedal H, Bergström U. ym.: initial observations of x-ray exposure and malignant transformation. Proc Natl fallow from the reactor accident at Chernobyl. Nature 321: Acad Sci USA 77: 7262-7266. 1980 192—193, 19S6 Munro T R: The relative radiosensitivity of the nucleus and cyto Fry F A, Clarke R H. O'Riordan M C: Early estimates or UK plasm of Chinese hamster fibroblasts. Radial Res 42: radiation doses from the Chernobyl reactor. Nature 321: 451—470, 1970 193-195, 1986 NCRP Report No. 46: Alpha-emilting Particles in Lungs. Na Hall E J, Het T K: Oncogenic transformation with radiation and tional Council on Radiation Protection and Measurements, chemicals. Int J Radial Biol 48: 1—18, 1985 Washington DC. 1975 ICRP Publication 23: Report of the Task Croup on Reference Rajewsky B, Franke Th, Groos E, ym.: »Heisse Teilchen». Lfn- Man. International Commission on Radiological Protec tersuchung radioaktiver Partikel aus dem Jahre 1961. tion, Pcrgamon Press, Oxford 1975 Alomprajtis 8: 3—24, 1962 ICRP Publication 26: Recommendations of the International Rytömaa T, Servomaa K: Radiation-induced IOTI/2 cell trans Commission on Radiological Protection. International formation is accompanied by enhanced expression of Commission on Radiological Protection, Pergamon Press, several different oncogenes. Abstract Book, p. 53, Euro Oxford 1977 pean Study Croup for Cell Proliferation, J 4th Meeting, Ox Kauppinen E, Hilfamo R, Aaltonen H. ym.: Radioactivity si2e ford 1986 distributions of ambient aerosols in Helsinki, Finland, dur STUK-B-Valo 44: Väliaikainen raportti. Säteilytilanne Suomes ing May 1986 after Chernobyl accident. Discussions of sa 26. 4.-4, 5. 1986. Säteilyturvakeskus, Helsinki 1986(a) accident scenario. Preliminary Report. Valtion teknillinen STUK-B-Valo45: Toinen valiaikaisraportti. Säteilytilanne Suo tutkimuskeskus. Tiedotteita 581. VTT, Helsinki 1986 messa 5. 5.—16. 5. 1986. Säteilyturvakeskus, Helsinki Kennedy A R, Fox M, Murphy G, ym.: Relationship between 1986 (b) Summary Rytömaa, T., Scrvomaa, K. and Toivonen, H.: CHERNOBYL REACTOR ACCIDENT AND RADIOACTIVE PARTICLES FOUND IN FINLAND Tapio Rytömaa, professori Kristina Servomaa, laboratorionhoitaja Harri Toivonen, TkT Säteilyturvakeskus, säteilybiologian laboratorio, PL 268, 00101 Helsinki Jätetty toimitukselle 4. 7. 1986 Hyväksytty julkaistavaksi 31.7. 1986 II TOIVONEN H., SERVONI AA K., RYTÖMAA T. Aerosols from Chernobyl: particle characteristics and health implications. In: von Philipsborn H. and Steinhäusler F., eds. Hot Particles from the Chernobyl Fallout. Theyern: Schriftenreihe desBergbau- und Industriemuseum, 1988; 16: 97-105. Aerosols from Chernobyl: Particle Characteristics and Health Implications Harri Toivonen, Kristina Servomaa and Tapio Rytömaa Finnish Centre for Radiation and Nudear Safety. P.O. Box 268, SF-00101 Helsinki, Finland Abstract the South and West and the fallout in Southern and Central Europe may therefore have contained ruthenium particles of The accident at Chernobyl reactor 4 created highly radioactive high activity. aerosols. X-ray analysis and scanning electron microscopy showed isolated single particles of chemically pure uranium A few weeks after the accident a contaminated Soviet train with readily measureabie activity coming mainly from Zr-95, carriage crossed the Finnish border. The dose rate was about Ce-141, Ce-144, Ru-103 and Ru-J06. Some of the small 1 mGy h near the outer surface. Extremely pure samples particles contained only ruthenium, but even these are chemi could be obtained from this source because panicles were cally uranium. In addition to the fallout samples collected from deposited on surfaces covered with oil or grease. The train Finnish soil, we also took samples from a contaminated Soviet must have been within a few hundred kilometres of Chernobyl, train carriage which was stopped at the Finnish border. because particles larger than 20 micrometer were found. The aerodynamic diameter of such particles is over 50 micrometer, The dose-distance relationship and direct observations of cell and they can stay airborne for a maximum of about five hours. cultures show that the high energy beta sources, Ru-106 and Ce-144, create a lethal area with a diameter of up to 1 mm for a panicle of 1000 Bq. Cell death then stimulates and sustains cell proliferation in the area surrounding the „Wack hole". This Particles in the Environment enhances the expression of malignant transformation, and the In southern Finland the activity concentration in the air was frequency seems to be extremely high compared with cultures 3 3 where the same total dose is evenly distributed over C3H/ high (200 Bq m of 1-131 and 10 Bq m of Cs-137) only on April 10T1/2 fibroblasts, 28, 1986. During the next day the concentrations decreased by two orders of magnitude (2). Similarly, most of the hot particles were deposited on the second day after the accident. Introduction Autoradiography of air filters showed that distinct hot spots An explosion or explosions occurred during the Chernobyl existed in the filters. The Finnish Meteorological Institute found accident. Because of this, part of the core (about 4%) was 17 particles with beta activity above 200 Bq in an air filter in mechanically broken (comminution) Some of the fragments Helsinki (3); the volume of air sucked through the fitter was were large (0.1-1 mm in diameter) but at least 1% of the 3500 rrP This measurement was made on April 28,1986. On contents of the core was converted to smaller particles that the following day only one particle was trapped in the sampling were transported over 20 km from the site of the reactor (1). system. The Finnish Centre for Radiation and Nuclear Safety This initial release moved towards the Nordic Countries. produced similar data: on April 28 there were about 80 hot Because of their settling velocity, particles larger than 8-10 spots in 1000 rrf of air; the beta activity of these particles was micrometer in physical diameter could not reach Finland, even more than 10 Bq one year after the accident (4). if the radioactive plume had initially risen to an altitude of two Man inhales daily about 30 m1 of air. Thus, during the early kilometers. fallout as many as one person in ten may have inhaled very The continuous graphite fire and increased temperature hot particles (> 200 Bq) in Ihe fallout area. Small particles that lasting for seven to ten days after the accident resulted in the must have been inhaled by everyone outdoors were much escape of a significant fraction of oxidized, volatile ruthenium. more abundant. However, obviously only a tiny fraction of this late release Later on, hot particles could be readily found in the environ reached Finland: the direction of the wind was then towards ment using a beta contamination meter. Small samples of B6?gb0u- und If0i/stwrtv96um THeuGrn B0i>ö 1&86 97 earth or surface plants, especially moss, containing unusual the reactor fuel. This behaviour is consistent with the observa radioactivity were identified and collected, and then subjected tion that some of the fuel fragments had lost variable amounts to autoradiography in the laboratory (Fig. 1). of ruthenium (Table 1). Whenever ruthenium was present in a particle together with cerium, and was not enriched, the ratio The activity of grass and moss and the number of hot spots Ru-t03/Ru-l06 gave the same bum up as the ratio Ce-141/ varied greatly in different parts of the country. Up to 10 000 Ce-144 (6). In general, however, the ratio Ru-103/Ru-106 is particles per square metre were detected in some open areas not a reliable measure of the burn up because the activity may (5); however, the mean deposition in Finland was less by a have originated from different fuel rods. factor of ten or more. The activity of these particles is about one Becquerel. Single inhaled particles of this activity cannot Almost all measured objects contained a few Becquerels of be detected by whole-body counting. volatile caesium. Most of the caesium was apparently simply Extremely hot particles were isolated from the contaminated present as an impurity in the sample but a small amount may Soviet train. The highest activity was measured in a particle also be condensed on the surface of the particles. Because of that contained 23 000 Bq of Ru-103 and 6 400 8q of Ru-106 the wide variations in fuel bum up, the expected isotope ratio Such particles were not airborne in Finland (6). Cf-134/ Cs-137 should vary between 0-1.2. The measured ratio, however, was quite constant (055 ± 0.09). This strongly suggests that the caesium is a mixture of atoms originating from different fuel rods. Particle Characteristics At low burn up (2500 MWdtU') plutonium forms about 0.1 % of The activity concentrations of the fission products in the reac the mass of the particle; at high bum up (20 000 MWd tU ) the tor fuel may be used to estimate the size of the individual concentration is about 0 5% The activity of plutonium and particles. The fuel of the RBMK reactor contained an activity of other actinides is only a tiny fraction compared with the activity 2 8 x 10" Bq tU' for either Zr-95 or Ce-141. These nuclides of cerium and ruthenium quickly reach their saturation level in the reactor core; the amounts do not depend drastically on the ft ^l bum up after a Scanning Electron Microscopy few months of irradiation The activity of Zr-95 and Ce-141 is Isolated samples were subjected to scanning electron micros- almost equal at moderate and high bum up of the fuel. The sopy (SEM). Initially, when we did not really know what to look activity of Ru-103 >n the core is slightly below that of Zr-95 or for under the microscope, it was difficult to identify a radioac Ce-141. tive particle among hundreds of tiny objects seen under the H a particle is a core fragment the activity of Zr-95 and Ce-141 microscope. The X-ray spectrum associated with SEM was, should be about 0.15 Bq for a particle with an effective however, most helpful for reliable particle identification. diameter of 1 micrometer (6). The activity is proportional to the A scanning electron micrograph of a fairly large hot particle, cube of the radius; thus, for example, a particle of 10 microme isolated from the Soviet train carriage, is shown in Fig.2. X-ray ter in diameter contains 150 Bq of Zr-95 and Ce-141. spectra from the individual grains and the whole conglomerate In principle there are five different types of uranium aerosol, indicate (Fig 3) that the particle is chemically pure uranium, i.e. distinguished by their different ruthenium contents: ruthenium that it consists of fragments of nuclear fuel. The architecture of may be in balance with the other fission products, or its the particle suggests that it could be mechanically broken into enrichment or depletion may be partial or complete compared smaller fragments. We did indeed observe this fragmentation with zirconium and cerium (Table 1) The nuclide ratios repre during the isolation of several apparently single particles, both sent the burn up of individual fuel rods. Because bolh zirco small and large (Fig.4). It is thus clear that radioactivity is nium and cerium remain fixed in the fuel fragments, the ratio of distributed between, and is present in, the small uranium the nuclides Ce-H1/Ce-144 and of Zr-95/Ce-144, can be used grains anti not as separate discrete entities. Interestingly, to estimate the bum up of the fuel (6). particles isolated from soil samples were nevar pure and the uranium grains were inseparably associated with contamina The observed ruthenium activity varies strongly between par tion material; occasionally, however, the exact location of the ticles, suggesting different aerosol formation mechanisms. In uranium could be seen directly (c>. Figs. 2 and 8) and, more the graphite fire UO was oxidized to U 0 . Similarly, ruthe ? 3 8 commonly, located by its X-ray spectrum. nium was oxidized to Ru04 or, possibly, to ruthenium carbo nate. The volatile ruthenium compounds escaped from Irie Some of the particles contain either too little or too much uranium matrix and condensed later on other tiny fragments of ruthenium compared with the amounts of zirconium and 98 cerium. We therefore isolated one apparently pure ruthenium than the annual natural background (Fig.6). If we were to particle for SEM to see whether this particle was a distinct estimate the chance of getting a single malignant focus in this entity and no longer associoated with the uranium fuel. The X- assay, using the transformation frequency seen after evenly ray spectrum revealed that even this particle consisted of distributed acute gamma radiation, more than 1000 test cultu uranium with no detectable quantity of any other element (6). res would be needed. Such a comparison is. however, unreali In general, the isolation of pure ruthenium particles for SEM stic for many reasons as indicated below. turned out to be particularly difficult for two main reasons: the particles are small in size and they stick extremely rapidly and In each of the three cultures the series of events was essen tightly to various surfaces, possibly through electrostatic tially identical. During the first days of the culture the cells forces. proliferated normally and a confluent monolayer was formed, achieving close contact with the hot particle. Subsequently, Radiation Doses however, cells near the radiation source accumulated a lethal dose and died (Fig.6). Around this zone, up to a distance of The average energy of the beta particles from Rh-106, the about 5 mm from the source, cell proliferation was active and daughter nuclide of Ru-106, is 1510 keV (78.8% intensity) and attempted to heal the small chronic wound (Figs6 and 7); 1220 keV (97.7% intensity) from Pr-144, the daughter nuclide these phenomena were readily established using time-lapse of Ce-144. A particle deposited in tissues irradiates a large cinematography (results not shown). In one of the three cultu volume, with diameters up to 3 cm (7). The radiation dose is res an apparent early transformed focus was spotted at two very high in the vicinity of a hot particle. Up to a distance of 0.5 weeks: a large focus, visible to the naked eye. was seen at mm the dose rate is above 1 Gy d' from a particle containing four weeks. In the other two cultures large transformed foci 1000 Bq of high energy beta emitters (Fig.5). At a distance of 5 were seen at 6 and 8 weeks, respectively. All foci were within mm the dose rate is still 1000 times higher than the back 2-4 mm of the radiation source. The culture morphology within ground exposure the three distinct areas indicated in Ftg.6 is shown in Fig.7. A There is not enough information on the delayed effects of scanning electron micrograph of one ot the particles surroun small, highly radioactive beta particles inhaled into the lungs ded by dead cells, is shown in Fig 8 (8). However, in animal experiments aerosols of CeOri44 seem to be more efficient in initiating lung tumours than Isolated transformed foci were cloned, expanded and analy plutonium (Pu02- 239) (9). zed for the activation of different oncogenes. These results will be reported in detail elsewhere, but it may be noted here that Particles of low activity are also potentially dangerous. A hot transformed cells revealed variable transcriptional activation of spot containing 100 Bq of Ru-106 or Ce-144 was fairly com several oncogenes, commonly c-fms, c-abl, and c-Ha-ras or c- mon in the fallout. The volume of cell death around such a Ki-ras. particle is small but by no means negligible (Fig.5). The radiation risk from the small particles remains to be defcm-ii- It seems clear that a unique, uneven radiation dose capable of ned. However, we should remember that from a micro dosi causing eel! death near the particle contributed to the expres metric point of view a point source is quite different from an sion of malignant phenotype in each culture. Around this death even distribution of activity: there are almost 100 000 radioac zone the cells received a relatively high sublethal dose and the tive decays each day from a particle with 1 Bq of activity and all probability of malignant transformation was therefore high. the energy is deposited near the source. Judging from the frequency 2.5 x 10' Gy' per surviving cell, seen after the Co-60 gamma dose, we can estimate that the Malignant Transformation occurrence of such a change was highly likely; the dose- distance relationship shows that the chance of inducing mali We have previously tested the ability of isolated hot particles to gnant transformation is high up to about 4 mm from the source cause malignant transformations of C3H/10T1/2 mouse fibro (dose proportional to r', number of cells in monolayer to f) and blasts in vitro (10,11). This cell line, commonly used in trans that this area contains enough cells to make it likely that such formation assays, shows malignantly transformed foci at a an induction does indeed occur, The indirectly induced and frequency of 2.5 x 10" per surviving cell after a dose of 1 Gy of sustained proliferation of these cells then results in a rapid external gamma radiation from Co-60 (11) Three particles, fixation of the genomic change and, therefore, promotes its isolated from soil and containing 1200. 400 and 300 Bq of Ce- expression and clonal development. 144 at the onset of the experiment, were sterilized by autocla- ving and then added to growing cultures. In this assay the We emphasize that, although low-level activity readily led to majority of cells receive an extra' radiation dose which is less malignant transformation in this study, the results may not 99 directly apply to man with inhaled fuel particles. Two factors report STUK-A-55. Finnish Centre for Radiation and Nuclear appear relevant in this context. First, it is unlikely that a Safety, Helsinki (1987) sufficiently active particle (large in aerodynamic diameter) 5) Rytömaa T., Servomaa K. and Toivonen H„ Hot particles could be fixed in the lungs so as to be able to cause a chronic from the Chernobyl accident: possible health hazard in Fin wound; thus the tumour-promoting effect would be lost. land. Duodecim 102,1190-1196 (1986) (in Finnish) Second, a small panicle possibly reaching the alveoli may not be active enough to resuft in significant ceil death, and thus the 6) Rytömaa T., Toivonen H, Servomaa K .Sinkko K and Kai- promoting effect would again be lost. A small, extremely active turi M., Uranium Aerosols in Chernobyl Fallout. Submitted for ruthenium particle may, however, be an exception. If such a publication (1986) particle is translocated to the lung-associated lymph nodes the 7) Cross W.G., Ing H., Freedman NO, Mainville J.. Tables of consequences could conceivabtv be hazardous. beta-ray dose distributions in water, air and other media. Chalk River Nuclear Laboratories, AECL-7617 (1982) 8) Mayneord W.V. and Clarke R.H.. Nature 259, 535-539 (1976.) References 9) Lundgren DL et ai. Toxis effects of repeated inhalation 1) IAEA Safety Series No 75 - INSAG -1. Summary report on exposure of Syrian hamsters to aerosols of Ce-144 or Pu02- the post accident review meeting on the Chernobyl accident. 239. In Biological implication of radionuclides released from International Atomic Energy Agency, Vienna (1986) nuclear industries. Vienna: International Atomic Energy 2) STUK-B-Valo. Reports 44 and 45. Finnish Centre for Radia Agency, IAEA-SM-237/41, (1979) tion and Nuclear Safety, Helsinki (1986) 10) Rytömaa T. and Servomaa K. Radiation-induced 10T1/2 3) Mattson R. and Hatakka J. Hot particles in air after Cherno cell transformation is accompanied by enhanced expression of byl accident. Finnish Association for Aerosol Research. Report several different oncogenes. Radiation Research (ARR/NRS series in Aerosol Science, No. 2, 28-30 (1986) (in Finnish) Abstracts) 107: 923 (1986) 4) Sinkko K, Aaltonen H, Mustonen R., Taipale T. and 11) Servomaa K , Ionizing radiation, malignant transformation Juutilainen J. Airborne radioactivity in Finland after the Cher and oncogenes. M.Sc. thesis. University of Helsinki (1987) (in nobyl accident in 1986, STUK-A-56. Supplement 1 to Annual Finnish) 100 Table 1 10 Type « Zr 10*RU »Ru '"Ce 144'C e Five different types of particles found in Finnish soil. Activities (Bq) at April 28, 1986*; see text. F 38 16 1.8 36 16 F- 15 3.5 - 11 7.9 F-- 7.8 0 0 10 3.7 F* 43 190 50 37 31 F+ + 0 170 33 0 0 ;a f b **f* « T* V Fig. 1 An autoradiogram (b) of moss (a). The exposure time is 2 d. The beta activity of the individual particles is about 1 - 10 Bq. 101 5W' .-> >.'&' &u7*P*'jil§rKrJ* ' 'I Fig. 2 Details of a large uranium particle (about 20 micrometer). Scanning electron micrograph showing the granular structure of the particle. Sintered uranium fuel has a different structure (larger granulae with sharp edges). This particle has obviously been in a molten state. 102 COUNTS ENERGY IKEV) Fig. 3 An X-ray spectrum of the particle shown in Fig.2. Uranium is the only chemical element that is shown to be in the sample. (Au comes from the sample preparation, and Cu and Zn from the sample tray). 103 Fig. 4 Autoradiogram of a large particle, spotted on the train carriage (see text), before and after a manual isolation process. Exten sive fragmentation of the initial particle is consistent with the architecture shown in Fig.2. Exposure time 1 min. # luBWttXJ HOI» Fig. 6 In vitro assay for malignant transformation by uranium partic les including graphic representation of the dose gradient in a realistic scale (c.f. Fig.5) Ft hot - particl* 1000 Bq Ä «•» - •v«n *l|r*»ut>© n 1000 9q/ < m' Fig. 5 Radiation doses from hot parttcles compared wilh even distri I bution of activity. A point source containing 1000 Bq of Ru-106 10 01 OS 1 or Ce-144 (and daughter nuclides Rh-106 and Pr-144) causes acute radiation effects in a volume of one milimetre in diame Dulanct. mm ter No beta burn is caused by even distribution. 104 Fig. 7 Light microscopic view of the cell culture, (a) A zone with dead cells adjacent to the radiation source (blacK spot in the centre), (b) Confluent monolayer of normal 10T1/2 cells far from the source, (c) Part of a malignant focus showing criss-cross architecture, multilayer of ceHs and mitoses. Fig. 8 Scanning electron micrograph of a hot particle surrounded by cell debris. The uranium is located on the top of the soil particle 105 Ill SERVOMAA K., RYTÖMAA T. Activation of oncogenes by uranium aerosols: an in vitro study. In: Brustad T., Langmark F., Reitan J.B., eds. Radiation and Cancer Risk. New York: Hemisphere (Cancer Series), 1989: 31-42. 4 Activation of Oncogenes by Uranium Aerosols: An In Vitro Study Kristina Servomaa Tapio Rytömaa Finnish Centre for Radiation and Nuclear Safety, Laboratory of Radiobiology Helsinki, Finland INTRODUCTION In the Chernobyl explosions and fire powderized nuclear fuel was released from the reactor core, causing an unexpected local fallout. Uranium aerosols, also transported by air to distant areas, contained nonvolatile fission prod ucts, including the 8 emitters *Zr, '"Ru, ^Ru, 141Ce, and "*Ce (Devefl et al. 1986; Rytömaa, Servomaa, and Toivonen 1986; Rytömaa et al. 1966; Toivonen, Servomaa, and Rytömaa 1988; Vin der Veen et al. 1986). These particles may pose a potential health hazard to humans dis tinct from acute 8 burns of skin and mucous membranes. One obvious hazard is long-term retention of a relatively small particle somewhere in the respiratory tract or in the lung-associated lymph nodes. An immobi lized uranium particle, containing several different fission products, cre ates a strong dose gradient around it, delivering a lethal radiation dose to adjacent cells and a high but sublethal dose to the surrounding cells (up to a distance of several millimeters or, depending on the particle activity, even centimeters) (Rytömaa, Servomaa, and Toivonen 1986; Rytömaa et al. 1986; Toivonen, Servomaa, Rytömaa 1988). The biological theory behind the suggested health hazard may also be associated with certain findings of radiation-induced malignant transforma tion in vitro as reported by Kennedy and colleagues (1980; 1984). These find ings are summarized as follows. If cultured cells (C3H 10T1/2) are irradiated 31 32 Conctf Risk from Low Dosos of Ionizing Radtotton whh a high acute x-ray dose and then allowed to grow to confluence and maintained for 4 to 5 weeks, a dose-dependent number of transformed malig nant foci arise. Transformed foci are expressed, per culture, at a frequency that seems to be constant (i.e., independent of the number of irradiated cells), at least over a certain range of cell densities. If the irradiated cells are subcul- tured (e.g., 10"4 dilution) after reaching confluence and then allowed to re- grow to confluence, however, the same constant number of foci develop on each new plate. Thus the risk (frequency of expression of malignancy) is increased 10 000-fold simply by subculturing. It is clear that a single uranium particle added to a test culture will deliver a low overall dose to the cells if averaged over the whole population. It appeared to us, however, that the strong dose gradient around the particle would produce a situation basically similar to the subculturing (i.e., stimula tion of growth) described above and, therefore, that a "hot particle" would probably be an exceptionally effective source of inducing malignant transfor mation in vitro. This chapter demonstrates that this is indeed the case: a hot particle produces transformed foci essentially in every culture. The activity of a reactor fuel particle need not be particularly high: a few hundred bec- querels, or less, of hard 0 activity (1<ÄRu or 14*Ce) is sufficient. In addition to measuring transformation frequencies per se, we also studied expression of several different oncogenes in the isolated malig nant cell clones. The results were compared with clones obtained by an evenly distributed high y dose (*°Co) and with clones forced to transform spontaneously. MATERIALS AND METHODS CEU CULTURES C3H 10T1/2 CL 8 mouse embryo-derived fibroblasts were maintained in Eagle's minimum essential medium with Earle's salts (EME) supple mented with 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 pg/ml). The cells were used in passages 9 to 15. A subconfluent cell culture was subcultured 1:4 on 25-cm* tissue culture flasks and irradiated 24 hours later (in experiments with uranium parti cles, irradiation was started at that time). TRANSFORMATION Cells were irradiated at room temperature with 3 Gy of "Co y rays at a dose rate of 0.4 Gy/min. After irradiation, the cells were trypsinized (0.1% trypsin) and suspended in complete medium, and 1 000 cells were plated 2 in tissue culture flasks (25 cm ). Cells were incubated at 37°C with 5% C02 in air and fed twice per week. After the cells reached confluence, the medium was replaced with EME containing 5% fetal calf serum. 33 Activation of Oncogtnes by Uranium Aerosols Hot partides (uranium aerosols) isolated from soil samples (Rytö maa, Servomaa, and Toivonen 1986; Rytömaa et al. 1986; Toivonen, Servo- maa, Rytömaa 1988) were sterilized by autodaving and then added to growing cultures. Particle activities are given in Table 4-1. After a 4- to 10- week incubation period, dense fod of transformed cells growing as a mul tilayer were easily distinguished morphologically from the flat confluent sheet of untransformed cells. Type III transformed fod of the C3H 10T1/2 cells (Reznikoff et al. 1973) were isolated, doned, expanded, and analyzed for the activation of different oncogenes. EXTRACT/ON OP RNA AND SLOT-BLOT ANALYSES Total cytoplasmic RNA was extracted from normal and transformed cells at the exponential growth phase by cell lysis in cold buffer with Nonidet P-40, phenol extraction, and ethanol predpitation (Servomaa 1987; Servo maa and Rytömaa 1988). RNA slot-blot analyses were performed with the isolated RNA in a dilution series (40, 20, and 10 /tg or 20, 10, and 5 pg). HYBRIDIZATION AND PROBES RNA filters were baked and then prehybridized and hybridized for 24 hours at 42°C in Denhardt's mixture (0.02% each of bovine serum albu min, Ficoll 400, and polyvinylpyrrolidone), 3 x standard saline citrate, 40% formamide, 50 mM hepes buffer, and salmon sperm DNA (150 pg/ml) to 32P-labeled nick-translated probes (specific activity > 108 counts/min per microgram of DNA). After hybridization the filters were washed (Servo maa 1987; Servomaa and Rytömaa 1988) and exposed on x-ray film with intensifying screens. The following cellular or viral oncogene probes were used: a 0.45- kilobase (kb) v-erb-A fragment (pAEPst0.45), a 0.5-kb y-fms fragment (pAEBamHI), a 0.45-kb v-fes fragment TABLE 4-1. Isolated uranium particle* used in the in vitro «May*. Activities of tome nuclides present in the uranium matrix arc given. Time of detection of transformed fod in the assay culture is also indicated. Total Appearance of Activity (Bq at 28.4.86) activity visible Particle at onset of foci number »Zr 106Ru H4Ce experiment (Bq) (weeks) 308 400 400 300 6 314 550 500 400 <9 301 1500 500 1150 1200 3 to 4 288* 4600 1100 3200 1200 10 *As a contaminant, this particle also contained 430 Bq of ,37Cs and 134Cs (at 28.4.86). 34 CoiKtf Risk from Low Dos*s of Ionizing Radiation ment, a 1.8-kb v-abl fragment, a 0.4-kb c-myc fragment (pML), a 0.45-kb c- Ki-ras fragment (p640), and c-Ha-ras oncogene (Rt). PHASE-CONTRAST AND SCANNING ELECTRON MICROSCOPY The cells were observed by an inverted phase-contrast microscope and photographed frequently. After each experiment, hot particles (uranium aerosols surrounded by cells) were examined in the Jeol JSM-35C scanning electron microscope. RESULTS With *°Co 7 rays (3 Gy) the transformation frequency was 2.5 x 10"* Gy~l per surviving cell under the assay conditions used. In 80 control cultures, no transformed foci developed. Expression of oncogenes in several differ ent malignant clones isolated from the irradiated cultures are shown later. Activities of four different hot particles used in the present study are given in Table 4-1. Scanning electron micrographs of two of the particles are shown in Fig. 4-1. The essential set-up of the experiments with the hot particles is out lined in Fig. 4-2. Each of the particles induced transformed foci in the assay culture in 4 to 10 weeks; the time of appearance of foci (visible to the naked eye) was not detectably associated with the particle activity (see Table 4-1). The foci were regularly seen within 2 to 4 mm of the radiation source; in one culture (particle 288 in Table 4-1) two additional foci were present about 10 to 15 mm away from the source. No apparent primary foci developed in the zero-dose area. Transformed and normal cell morphologies in the two nonlethal radi ation zones indicated in Fig. 4-2 are shown in Fig. 4*3. A revealing culture architecture around a hot particle is shown in Fig. 4-4. It is important to note that the wound did not develop immediately: the cells first produced a confluent layer adjacent to the particle but began to die after a few days (see Fig. 4-4). The wound was then maintained by the constant high dose delivered to the incoming cells. These simple observations speak against a release of any toxic chemical from the source-containing sod. Around the chronic wound, mitotic activity of the C3H 10T1/2 cells remained high throughout the entire follow-up. Often several mitoses were seen in a single visual field. Judging from cell morphology and mi totic activity, the location of a transformed focus was sometimes guessed at an early stage; often, however, a macroscopic focus finally appeared at a location that was not suspected during the follow-up. This was evidently caused by repeated handling of the cultures (e.g., for microscopy), which occasionally also dislodged some loosely attached transformed mitotic cells to distant areas (culture with particle 288). 35 Activation of Oncogenes by Uranium Aerosols FIGURE 4-1. Scanning electron micrographs of two uranium particles (301 and 314 in Table 4-1) photographed after the in vitro assay. A layer of dead cells is seen in the background. The location of uranium within the soil particle is shown by the arrow. Expression of several different oncogenes in the radiation-induced transformed foci (isolated clones) is compiled in Table 4-2. Some examples of the slot-blot analyses of transcript levels are shown in Fig. 4-5. In the spontaneously transformed clone 51 (Table 4-2 and Fig. 45) the c-myc oncogene was amplified; this transformed cell line also contained numer ous double-minute chromosomes (not shown), which is quite common to 36 Cancer Risk from Low Dos«s of Ionizing Radiation tumor cells with amplified c-myc genes (Alitalo et ai., 1985). Judging from the intensity of the c-Ha-ws expression in the other spontaneously trans formed clone S2, the activation was probably also by gene amplification (not confirmed). Note that these spontaneous transformations were ob tained by long-term repeated subculturing (more than 15 passages) of the C3H 10T1/2 cells. In contrast to the apparent gene amplification in the two spontane ous transformations, the radiation-induced malignant cell lines showed highly variable oncogene expression patterns. The two cell lines induced by hot particles and studied thus far display grossly similar expression patterns (Table 4-2), however, which may not have arisen by chance. Changes in the transcript levels alone do not allow far-reaching conclu sions to be made about the radiation-induced oncogene activations or their relation with the malignant phenotype. DISCUSSION The present results show that Chernobyl-released hot particles are effec tive in inducing transformed foci in C3H10T1/2 cells in vitro. For all prac tical purposes, malignant foci seem to develop in every culture and appear at the same time or only a little after an evenly distributed acute high dose of "Co 7 radiation. Furthermore, in all cultures foci developed near the particle (usually within 2 to 4 mm), and none arose directly in the essen- ^^< » 55 JO / N^ *f fane*, i*m 7 n*ö*%» MtMrfOf* f uOtoffMt «««• FIGURE 4-2. The experimental set-up 37 Activation of Oncogene» by Uranium Aerosoli FIGURE 4-3. (A) Normal C3H 10T1/2 cell layer (no-dose area in Fig. 4-2) and (B) part of a transformed focus (cell line 314 in Table 4-2). Inverted phase-contrat microscopy (x700). tially zero-dose area (see Fig. 4-2). In the unirradiated control cultures (passage 15 or less) not a single transformed focus developed (80 cultures in total). It is also worth noting that, judging from the 7-irradiated cul tures, sensitivity (of the cell line used by us) to radiation-induced transfor mation (2.5 x 10~4 Gy~f per surviving cell) was essentially identical to that reported by others (Hill, Han, and Elkind 1987; Terzaghi and Little, 1976). The two spontaneously transformed foci (studied for oncogene ex pression; see Table 4-2) were obtained by long-term repeated subculturing (tens of passages). In these foci the dominant change in oncogene expres sion appears to be different from that of the foci induced by radiation and seems to involve gene amplification. 38 Cancer Risk from Low DOMI of Ionizing Radiation It is not possible to prove unequivocally that, in the present experi ments, initiation and promotion of malignant foci were both directly caused by radiation (rather than being spontaneous and caused by pro longed mitotic stimulation alone). Clearly, however, the high expression frequency was associated with the uneven radiation dose delivered by the hot particles and mediated through the growth stimulation caused by the chronic wound. In this context, we omit detailed discussion of our results but present the following conclusions that we find to be justified: 1. The Chernobyl-released uranium particles are extremely effective in producing expression of malignant transformation in vitro. In terms of an average cell dose over the whole population, the point source is FIGURE 4-4. Hot particle (298 in Table 4-1; black dot in micrograph surrounded by OH 10T1/2 cells.) At 3 days the radiation dose has not yet been lethal to the cells; later a chronic wound develops around the particle and is maintained in spite of continuous influx of (sub- lethally damaged) new cells (x 1 400). TABLE 4-2. Summary of oncogene transcript levels in the untransformed and some malignantly transformed cell lines (total/mRNA, isolated from cytoplasm). Cell lines SI and S2 were forced to transform spontaneously by extensive passage; cell lines 77a to 77f were induced by evenly distributed y radiation ("Co; 3 Gy) and cell lines 301 and 314 by a point source (uranium particle). A minus sign does not necessarily indicate total absence of the transcript; it indicates the control level. Plus signs indicate differences between the parent (C3H 10T1/2) and transformed cell lines (see Fig. 4-5). Oncogene Expression Cell Line myc myb Ha-nas Ki-ras erb-A fms fps abl sis nf WTV2 - - 51 + + 52 ++-- - - + 77a 77b ++ - 77c - + + + ( + ) + 77d + + + + 77e - - - 77f - 301 + + + 314 + + + - + o fms myc »OTl 2 10Tl 2 S2 S2 77» 77a 77C 77d 77B 77e 77c 77e SI 314a 314b FIGURE 4-5. Examples of slot-blot analyses of c-myc and c-/ms transcript levels in the untransformed and some transformed cell lines (see Table 4-2); 314a and b refer to two different clones isolated from the same culture. 41 Activofion of Oncogenes by Uranium Aerosols orders of magnitude more effective than the evenly distributed 7 radia tion. 2. The major factor responsible for this effect is focus promotion caused by the wound-mediated permanent increase in cell proliferation among the population receiving a high but sublethal radiation dose. This result is strongly reminiscent of the situation in which a similar effect is ob tained by subculruring (Kennedy, Cairns, and Little 1984; Kennedy et al. 1980). 3. Expression of malignant transformation in this assay is not related to radiation dose in the conventional way (such as mean dose to the whole population). Also, there is no obvious difference between the transformation efficiency (or rather probability of its expression) of par ticles with different amounts of activity. Some of these and other con clusions appear to be trivial to the assay system used here, but the conclusions are not necessarily trivial if applied to conditions in vivo. For example, one may vary the size of the culture flask (i.e., the mean radiation dose to the tissue) without any effect on the transformation frequency (see Fig. 4-2). 4. Expression of oncogenes in different radiation-induced malignant foci varies from focus to focus. The pattern may be somewhat more con stant in foci developing after uneven radiation dose (foci arising pri marily by the promotion of expression) and in foci forced to arise spon taneously. 5. Inhaled uranium particles are less dangerous in vivo than what may be suggested by the present in vitro assay. The reasons are that the parti cles are unlikely to be permanently immobilized in the body (so that there would be no production of chronic wound) and that, if they are (e.g., in alveoli or in lung-associated lymph nodes), activities of these necessarily small particles would not be high enough to produce con tinuous, sufficiently large-scale cell loss (wound). Potentially the most dangerous particles would be those that are small and heavily enriched in ruthenium (Rytömaa et al. 1986; Toivonen, Servomaa, and Rytömaa 1988). REFERENCES Alitalo, K., K. Saksela, R. Winqvist, R. Alitalo, ). KeskiOja, M. Laiho, M. Ilvonen, S. Knuutila, and A. de la Chapelle. 1985. Acute myelogenous leukemia with c-myc ampli fication and double-minute chromosomes, lanat 2:1035-39, Devell, L., H. Tovedal, U. Bergström, A. Appelgren, J. Chryssler, and L. Andersson. 1986. Initial observations of fallout from the reactor accident at Chernobyl. Nature (London) 321:192-93. Hill, C. K., A. Han, and M. M. Elkind. 1987. Promotion, dose rate, and repair processes in radiation-induced neoplastic transformation. Radial. Ret. 109:347-51. Kennedy, A. R., J. Cairns, and J. B. Little. 1984. Tuning of the steps in transformation of C3H 10T1/2 cells by ^irradiation. Nature (London) 307:85-86. Kennedy, A. R„ M. Fox, G. Murphy, and ). B. Little. 1980. Relationship between x-ray 42 Cancer Risk from Low Doses of Ionizing Radiation exposure and malignant transformation in C3H 10T1/2 cells. Prvc. Natl Acad. Sri. U.S.A. 77::_^-66. Reznikoff, C A., J. S. Bertram, D. W. Brankow, and C. Heidelberger. 1973. Quantitative and qualitative studies of chemical transformation of clone C3H mouse embryo cells sensi tive to postconfluence inhibition of cell division. Cancer Res. 33:3239-49. Rytömaa, T, K. Servomaa, and H Toivonen. 1986. Chernobyl reactor accident and radioac tive particles found in Finland (in Finnish), Duodecim 102:1190-%. Rytömaa, T., H. Toivonen, K. Servomaa, K. Sinkko, and M. Kaituri. 1986. Uranium aerosols in Chernobyl fallout. Nature {London) submitted for publication. Servomaa, K. 1987. Ionizing radiation, malignant transformation and oncogenes (in Finnish). Pro gradu study. Department of Genetics, University of Helsinki. Servomaa, K., and T. Rytömaa. 1988. Suicidal death of rat chioroleukemia cells by activation of the long interspersed repetitive DNA element (LIRn). Cell Tissue Kinet. 21:33-43. Terzaghi, M, and J. B, Little. 1976. X-radiation-induced transformation in a C3H mouse embryo-derived cell line. Oncer Res. 36:1367-74. Toivonen, H., K. Servomaa, and T. Rytömaa. 1988. Aerosols from Chernobyl: Particle charac teristics and health implications. In Hot particles from the Chernobyl fallout, ed. H. von Philipsbom and F. Steinhäusler, pp. 97-105. Schrifrenreihe der Bergbau-und Indus- triemuseum Ostbayem Theuern: 8 and 16. Van der Veen, J., A. van der Wijk, W. C. Mook, and R. I. deMeijer. 1986. Core fragments in Chernobyl fallout. Nature (londan) 323:399-400. IV SERVOMAA KM RYTÖMAA T. Malignant transformation of mouse fibroblasts by uranium aerosols released from Chernobyl. In: Riklis £., ed. Frontiers in Radiation Biology. Weinheim: VCH, 1990: 589-594. Malignant transformation of mouse fibroblasts by uranium aerosols released from Chernobyl KRISTINA SERVOM AA AND TAPIO RYTÖMAA Laboratory of Radiobiology, Finnish Centre for Radiation and Nuclear Safety, P. 0. Box 268, SF-00101 Helsinki, Finland In the Chernobyl explosions and fire, powdcrized nuclear fuel caused an unexpected local fall-out. Uranium aerosols, also transported by air to distant areas, contained all nonvolatile fission products, including several hard beta-emitters. We have isolated these particles and considered that they may pose a potential health hazard to man, distinct from acute beta bums of skin and mucous membranes seen in the rescue personnel. We have isolated these particles from different sources, including surface plants in Finland (Rytömaa, Servomaa, Toivonen, 1986) (Fig. 1) and contaminated Russian vehicles stopped at the Finnish border. Some of the nuclear fuel particles isolated by us from a Russian train carriage were quite large (physical diameter up to 0.1 mm) with maximum beta activity of about 100 kBq (^Zr, ,41Ce, I44Ce, ,03Ru and 106Ru; note that volatile fission products, especially 134Cs and ,37Cs, are not present in these particles). In the early fall-out, i.e. before the decay of several short lived fission products, activity of the particles has been an order of magnitude higher. Large particles cause an acute beta bum of skin and mucous membranes within minutes; such burns were common among the rescue persinrd in Chernobyl. Physical characteristics of the fuel particles isolated by us are described in some detail in Rytömaa et ai. (1986) and Toivonen et ai. (1987). We considered (hat even moderately active uranium particles may pose a potential health hazard to man, distinct from acute stochastic effects. One obvious mechanism is based on a long-term retention of a sufficiently small but active particle somewhere in the respiratory tract or in the lung-associated lymph nodes. An immobilized uranium particle creates a strong dose gradient around it, delivering a lethal radiation dose to adjacent cells and a high but sublethal dose to the surrounding cells. 590 r . \ m * & J Figure 1: Small sample of moss (A) showing numerous uranium particles in an autoradiogram (B). This sample was collected from an open area in western Finland, i.e. about 1,000 km away from the Chernobyl reactor, and it suggests that in many places in Europe chances of inhaling similar particles must have been high at the time of particle fall-out. All of the particles seen in this figure were, however, relatively weakly active (<10 Bq) and would probably not cause the effects described in the present paper. We have studied this situation in an in vitro assay, using C3H/10T1/2 cells (American Type Culture Collection, Rockville) as the target tissue. An example of the radiation-induced cell death near an immobilized uranium particle is shown in Fig. 2. Adjacent to the chronic wound, mitotic activity is high and sustained because of the wound-stimulated physiological cell regeneration. On the other hand, these rapidly proliferating cells are also subject to an essentially maximal risk for malignant transformation by radiation because they will eventually receive the highest possible but still sublethal radiation dose. One would therefore expect that the long-lasting exposure acting simultaneously as a cancer initiator and promoter results in a high probability for the expression of malignantly transformed foci. Experimental results show that this is indeed the case. In every culture studied by us one or more malignant foci developed in 4-8 weeks, usually 2-4 mm away 591 from the radiation source. In unirradiated cultures (80 in total) not a single focus developed, nor were transformed foci seen in any of the test cultures in areas that were well outside the regeneration zone (because of the relatively short range of beta particles, radiation dose becomes 'negligible' at about 10-20 mm from the source). Figure 2: Uranium particle (black spot) surrounded by a chronic radiation-induced wound Noie the sustained influx of C3HH0TK2 cells to the wound area from the surrounding cell zone. The size of the wound area varies with particle activity but the general view is always the same (lowest activity tested 300 Bq). Inverted phase-contrast microscopy, magnification 700x . Using scanning electron microscopy (SEM), very early stages of the development of transformed foci could be easily spotted within the critical zone. Examples of the initial focus progression are shown in Figs, 3-S; for comparison, SEM picture of a fully developed, type III transformed focus (also seen by naked eye) is shown in Fig. 6. We have isolated, cloned, and expanded some of the malignant foci induced by uranium panicles. From these cultures expression of several different oncogenes was studied and compared, besides nontransformed C3HIWT1/2 cells, to oncogene Figure 3: Scanning electron micrograph of a large uranium particle, surrounded by a layer of dead CJ//77077/2 cells. This particle was isolated from a contaminated Russian train carriage, and its beta activity, at the end of April. 1986, was about 100 kBq. At the time of the in vitro assay, radiation dose rate at the extreme border of this visual field was over 10 Gy per hour. Figure 4: S EM photograph of an apparent early transformed focus about 5 mm away from a uranium particle. At this distance the beta dose rale from 1MRu and l44Ce was 0.1-0.2 Gy per day and hence the cells have accumulated a total dose of about 3 Gy during the 3-week culture period of this assay. Typical criss-cross morphology of a type III focus is emerging. In the right lower comer an adjacent monolayer of untransformed cells is seen. 593 Figure S: SEM photograph of another apparent early focus, a few mm away from the radiation source. Malignant transformation in this area is suggested by mitotic divisions on top of a continuous monolayer (note thai one of the daughter cells is already flattening and piling up of cells must follow), and by the presence of abundant intracytoplasmic vesicles in some flattened cells (characteristic to transformed C3H/10T1I2 cells (see Lloyd and Henning, 1983). Figure 6: SEM photograph of a large (visible to the naked eye) transformed focus, again a few mm away from a uranium particle. The picture was taken from the central area of the focus and hence the criss-cross morphology typical to the edges is less distinct; on the other hand, thickness of the focus and its high cellularity arc apparent, 594 expression in foci induced by evenly distributed ^Co gamma radiation and also to a few foci 'forced' to develop spontaneously, i.e. tends of passages). These results, described in more detail in ref. 4, suggest that enhanced expression of c-Ha-ras. c- Ki-ras. c-fms and c-abl may be relatively common in transformed cells induced by uranium particles. In conclusion, we feel that the potential health hazard arising from nuclear fuel particles, especially if immobilized in alveoli or in lung-associated lymph nodes in man, must be taken seriously, and should be carefully evaluated in future epidemiological studies. This potential hazard does not evanesce by conventional but biologically unrealistic dose equivalent calculaiions although, of course, (he present n vitro findingsma y not directly apply to an in vivo situation either. References Lloyd, E.L. and Henning, C.B. Morphology of cells malignantly transformed by alpha particle irradiation. In: Ullrastructural Effects of Radiation on Tissues and Ceils, pp. 35-40, 1983. Scanning Electron Microscopy, Inc., AMF O'Hare. Rytömaa. T., Servomaa, K.. Toivonen. H. 1986. Hot panicles from the Chernobyl accident: possible health hazard in Finland. Duodecim 102:1190-1196 (Finnish). Rytömaa, T., Toivonen, H.T Servomaa, K.. Sinkko, K.. Kaituri, M. 1986. Uranium aerosols in Chernobyl fallout. Submitted to Nature. Servomaa, K„ Rytömaa, T. Activation of oncogenes by uranium aerosols: an in vitro study. Proc. Nordic Cancer Union Symp. 1987: Radiation and Cancer Risk (Hemisphere, Washington, in press), Toivonen, H., Servomaa, K., Rytömaa. T. Aerosob from Chernobyl: particle characteristics and health implications. Proc. Slh Radiometric Seminar Theyern, West Germany, 1987 (in press). V RYTÖMAA T., SERVOMAA K. Identification of a putative growth inhibitor gene in rat chloroleukaemia cells. In: Baserga R„ Foa P., Metcalf D., Polli E.E., eds. Biological Regulation of Cell Proliferation. New York: Raven Press, 1986: 93-101. Identification of a Putative Growth Inhibitor Gene in Rat Chloroleukaemia Cells T. Rytömaa and K. Servomaa Finnish Centre for Radiation and Nuclear Safety, Laboratory of Radiology, Box 268, SF-00101 Helsinki, Finland Rat chloroma, induced by 3-methylcholanthrene (fl), is a highly malignant myeloid leulcaemia, which has been maintained in our laboratory for 25 years. For the last 10 years these cell3 have been propagated in suspension culture in vitror they grow rapidly (10) in many types of culture media, including chemically defined media supplemented with low concentrations (5 yg/ml) of insulin and transferrin. \/hen chloroleukaemia cells reach a certain medium and cell-line dependent critical population density, cell proliferation rate decreases rapidly and, later, cell death begins to occur (Fig. 1). The mechanisms for the inhibition of cell proliferation and the subsequent cell death are not immediately ob vious. However, trivial explanations, such as shortage of essential nutrients and growth factors, seemed to be excluded. For instance, subcultures commonly fail if the population is allowed to bypass the critical density, in spite of a subsequent dilution of viable-looking cells into a fresh, growth- supporting medium (see also Fig. 1). "hese findings suggested to us that at the critical population density the chloroleufcaemia cells are "reprogrammed", i.e. that they are triggered to slow down the growth rate (and subsequently to die) . We thought that if this was the case then it should be possible to identify the activated gene associated with the growth inhibition. 94 GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA FIG. 1 Growth of chloroleukaemia cells in suspension culture supplemented with 1 % foetal calf serum. In this medium the critical population density, for the cell line used here, is reached at about 4 x 10^ cells/ml, because the cells, apparently blocked in the G\ phase of the cell cycle, do not enter mitosis normally, increase in the cell count slows down 9-10 h later. Dying cells begin to appear after additional 10-15 h (at the peak population density, about 10 % of the cells are dead by morphologi cal criteria; mitotic figures are still present, but relative ly rare). Medium change shortly before or after the critical density allows some further slow>growth, and delays cell death by about 24 h, but does not rescue the population. Black arrows indicate points studied in the screening of the multicopy-gene transcripts (see laturi. One of the putative growth inhibitors of chloro- leukaemia cells is granulocytic chalone (2,"T,11). However, the native inhibitor has not been identi fied unequivocally and we therefore thought - even if this inhibitor was involved here - that synthesis of oligonucleotides corresponding to the synthetic inhibitory pentapeptide (1,6) may not be useful in the gene identification. We speculated that another approach might be successful. The first important finding was that chloroleu kaemia cells contain strongly amplified DMA sequences (Fig. 2). It was, of course, clear that a multicopy gene seen in a highly malignant, established leukaemia cell line was more likely to be an oncogene than a hypothetical gene associated with growth inhibition. Attempts were thus made to identify the amplified sequences in the chloroleukaemia cells by hybridisa tions using 15 different cloned oncogene probes (v- src, v-abl, v-fps/fes, v-erbB, v-fms, v-raf, c-mos, v- sis, c-Ha-ras, c-Ki-ras, N-ras, c-myc, rT-myc, v-mv>>, v-erbA); all probes failed to show detectable homology with the amplified sequences (many of these oncogenes are expressed in chloroma cells, however; results not shown). Further experiments with additional oncogene probes were considered unnecessary at this stage of the study. GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA 95 B M E B/H B/E M/E t/m FTG. 2 Amplified restriction fragments in chloroleu kaemia DNA cut with endonu- cleases BamHI (B), HindllT (H), EcoRI (T.) , and their combinations. After *-5.i electrophoresis in 1 * ^,S5 agarose gel, the amplified fragments are readily seen *~2-2 when Southern blots (9) are hybridised with BamHI- "*" "M digested nick-translated ehloroleukaemia DNA, and 1 autoradiographed. The fragment sizes were estimated from co-electrophoresed * DNA markers. Chloroleukaemia DITA cleaved with different restriction endonucleases thus showed several strongly amplified DNA bands. These restriction fragments, isolated from the agarose gel, provided a set of probes, obviously specific for the amplified gene. Using this set we then screened for multicopy-gene transcripts in chloroleukaemia RITA, isolated before and after the time point when the cell population density becomes critical (cf.Fig. 1). The results showed that expression of the multicopy gene increased by a factor of about 10 before the detectable growth inhibition (Fig. ?). Two of the restriction fragments (the 5-kb BamHI fragment and the l.?5-kb FcoRT fragment) v/ere then cloned into pBR322 and pBR32fl, respectively. The desired recombinant clones were identified using the set of isolated restriction fragments as probes and further confirming the indentity of the selected clones by endonuciease mapping. Mote that a prelimi nary restriction map had been obtained from results such as shown in Fig. 2. Using the cloned genomic fragments as new probes we then restudied the expression of the amplified gene. The results confirmed that transcripts are present already during the exponential growth phase, but that this does not yet inhibit the overall growth rate markedly. Tt may be noted, however, that time-lapse photography of rapidly growing chloroleu kaemia cells showed that occasionally individual cells are reversibly blocked in the cell cycle, probably in the G\ phase as judged from the cell size. This blockage is not always caused by an irre- % GROWTH INHIBITOR GENE IN CHLOROLEUKEM1A A B ti: zzr FIG. l Expression of the multicopy-gene in rapidly growing and in inhibited rat chloroleukaemia cells. A Riot-blot analysis of total RNA, isolated from chloroleukaenia cells in the exponential growth phase (upper two lines; serial dilutions of 16 and 48 ug RITA, respectively) or from the cells collected after reaching the critical population density (lower two lines; serial dilutions of 12 and 24 ng RITA). The probe used was the smaller EcoRI restriction fragment of the amplified sequence, nick-translated with *2p-(\C?P. B To further control for the added amounts of RNA, the filters were later hybridised with cloned oncogene and chloroma gene probes (in the example shown a cloned but functionally unidentified chloroleukaemia gene fragment was used). versible difficulty in the cell-cycle progression because, after a spontaneous release of the blockage, the whole progeny of the temporarily inhibited cell proliferated at the normal fast rate. It may also be noted that death of the inhibited cells, or any of the rapidly proliferating chloroma cells, was extremely rare during the exponential growth phase. Probing with the cloned gene fragments then specifically confirmed that expression of the multicopy-gene is rapidly activated when the cell population reaches the critical density (Fig. 4). According to preliminary analyses of Northern blots, several differently sized transcripts are formed, ranging from -0.6 to -10 kb (results not shown). Surprisingly, preliminary findings then suggested that the activation of the gene expression is associated with further amplification at the genomic level (results not shown). This observation would make it understandable, however, that down-regula tion of the gene action is not easy; as already noted, the effects can not be reverted by diluting the population density shortly before visible cell death or by providing to the cells new, fresh medium. GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA 97 An PIG. 4 Expression of 40 20 10 the amplified gene in chloroma cells versus «ft ' population density 69 ~ (cf.Fig.l). Cell «n mm- -• counts (x 10"^ per ml) are shown on the left; 410 •"•" -- tbe blotted amounts (pg) ^^^ of total (cytoplasmic) RNA 510 MmW mm* *—• are given above the slots. The probe was the 2.2-kb EcoRI fragment of the cloned 5-kb BaroHI fragment, isolated from the plasmid pCB4. Five to 10-fold increase in the transcript level, as estimated by densitometric scanning of the autora- diograms, was seen to occur in less than 4 h (note population growth from 410 to 510 x 10^ cells per ml with Ta = 11 h) . There was some interest to see whether or not the strong amplification of the chloroleukaemia gene present in all of our current sublines was a result of the long-term in vitro propagation of these cells. Analysis of DNA from cell lines after long- or short-term culture indicated that similar gene amplification had obviously been present in the chloroma cells already during the in vivo maintenance (Fig. 5). Insight into the structural and functional aspects of the gene and the putative gene pro.-luct is not obvious from the results presented thusfar. Because we felt that fruitful sequence analysis should start with cnrTA rather than amplified genomic sequences, we established a cDNA library using poly( A )+Rr7A isolated from chloroma cells expressing the amplified gene maximally, i.e. after reaching the critical population density. Using the cloned gene fragments as probes we have now identified cDHA clones for further analyses, but sequence data are not yet available. In the meantime, hov/ever, another experimental approach has resolved the nature of the growth inhibitor gene. Search for this evidence began with the observa tion that the cloned probes hybridised strongly with the continuum of differently sized chloroma DITA fragments under conditions of high stringency (note the "background" in Fig. 5). It was thus clear that the probes contained sequences homologous 98 GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA FTG. 5 Presence of amplified sequences in different chloroleukaemia cell lines. Two of the cell lines were propagated in suspension culture for relatively short periods of time, about two months (line E*) or about a year (line E). The cell line C was established from a local chloroma tumour in years ago? it has been maintained in vitro since then. Southern blot of Hindlll-cut DHA (4 or G \i q DHA per lane) was hybridised with pCB4. Mote that DHA of Chinese hamster ov^ry cells (CHO) does not hybridise with this probe under conditions of high stringency. to an interspersed repetitive element in the chloro leukaemia cell genome and that direct identifi cation of this element could be possible without conventional sequence analysis. The size of the 1.35-kb EcoRI fragment of the chloroma DHA in the recombinant plasmids suggested to us that this ubiquitous chloroma sequence could be homologous to the discrete 1.35-kb EcoRI band seen in the mouse genome, i.e. to an element found in retrotransposons (4). To test this possibility we isolated D1TA from 3T3 and 10T1/2 mouse fibroblasts and cut the DITA with EcoRI. Electrophoresis through an agarose gel showed, as expected, the discrete 1.35-kb band after ethidium bromide staining. Hybridisation of this band with the 1.35-kb chloroma DNA fragment gave a positive signal under conditions of high stringency (Fig. 6), thus indicating that the 1.35- kb EcoRI fragment of the rat chloro.ua genome is strongly homologous to a retrotransposon sequence in the mouse DITA. Comparison of the restriction maps of Llffd (ref. 4) and pCB4 (Fig. "M suggested further that the q^no in the chloroma cells, associated with th^ growth inhibition, is not only homologous to the 1.35-kb EcoRI fragment of the mouse retrotransposon but that it apparently contains a similar open reading frame. Tt is thus interesting to note that the putative Mild gene product,possibly * DNA-binding protein with do- GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA 99 FIG. 6 Homology between the 1.3 5-kb EcoRI fragment HUMAN ion 2 STS 0f a mouse retrotransposon « 2 a 2 42 (3T? and iOTl/2 fibroblasts) and the 1.35-kb EcoRI fragment of chloroma DNA. The probe was isolated from a cloned 5- kb BamHI fragment of the multicopy-gene (pCB16), nick-translated (0.6 x 108 cpm per yg), and hybridised (l.^S x 106 cpm/ml) under , stringent conditions with •• • "•* *"115 EcoRI-cut mouse and human (blood cells) DNA (Southern blot after electrophoresis through O.fl % agarose, 2 or 4 ug DNA per lane; exposure time 3 days). FIG. "» Restriction map of • m r*t •«• * pCB4, compared with a clone of ' 'UtiJiliätf1"' mouse retrotransposon (4). The rti • t tM • * partial maps are aligned with 1 tTrMt ' respect to the 1.35-kb EcoRI ! ! ' *"'• * * ! fragments (shaded boxes). ORF 1 ' *"**" ' \lut = open reading fr*me, in wi^ „„ |t*. kilobases, of i,lMd; * « = repetitive promoters. Restric tion sites of pCB4 are shown for BamHI (B>, HindHI (H), EcoRI (E), PstI (P) and Bglll (G). Note that individual clones of pCB (and also of LIMd) are polymorphic for several restriction sites. mains homologous to several reverse transcriptases (4), also contains many cryptic peptides flanked with r.v/o basic amino acids (i.e. that these sequences could be bioactive peptides; see e.g. refs. 3,5). Although it is too early to speculate in detail of direct functional connections of the mobile genetic element with the observed growth inhibition, it is nevertheless tempting also to suggest that the apparent further amplification means that the gene sequences could kill the cells (and also inhibit proliferation) more directly, intriguingly by being suddenly retroposed into damaging locations in the chloroleukaemia cell genome. 100 GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA NUMMARY The present results show that after a critical population density the growth rate of cultured chloro leukaemia cells is strongly inhibited and the cells later enter a non-viable state. Interestingly, these effects are caused by a sudden activation of the major long interspersed repetitive element (retrotransposon) in the cell genome. Gene transcript level is increased about 10-fold shortly before the onset of growth inhibition and this increase seems to be associated with further gene amplifica tion. It is tentatively suggested that the growth inhibition is mediated by cryptic bioactive peptide(s) released from the putative gene product and that the subsequent cell death is generated by a unique retroposition of the gene sequences into critical locations in the cell genome. REFERENCES 1. Foa, P., Maiolo, A.7., Lombardi, L., Ciani, A., Chillemi, P., and Pölli, E.E. (19A3): In: IV International Symposium on Chalones, edited by L.A. Piruzyan and G.M. Elbakidze, pp. 12- 16. Moscow. 2. Foa, P., Maiolo, A.7., Lombardi, L., Rytömaa, T.,and Polli, E.E. (1982): Hcand. J. Haemat., 29:25"'-264. T. Gimenez-Gallego, G., Rodkey, J., Bennett, C, Rios-Candelore, M., DiHalvo, J., and Thomas, K.(19fl5): Science, 230: l^fl5-1.3ftfl. 4. Loeb, D.P., Padgett, R.U., Hardies, 5.C.,fThehee, 17.R., Comer, M.B., Edgell, M.H., and Hutchison IIT, C.A. (19R6): Mol. Cell. Biol., 6:16fl- 1R2. 5. Mason, A.J., Hayflick, J.f!., Ling, M., Esch, F., Ueno, N., Ying, fl.-Y., Guillemin, R., Miall, H., and fieeburg, P.H. (19R5): Nature 318:659- 663. 6. Paukovits, U.R., and Laerum, O.D. (1904): Hoppe-feyler's Z. Physiol. Chem., 365:303- 311. GROWTH INHIBITOR GENE IN CHLOROLEUKEMIA 101 Rytömaa, T., and Kiviniemi, K. (l.*>f;o): nature, 222: 995-9<>fi. Shay, H., Gruenstein, M., and Harris, Ow(1955): Acta Haemat., 14: S:*'»-:^. Southern, F.M. (1975): J. MoJ . Biol., 98:503- 517. Toivonen, H., Poa, P., Paile, W., and Rytömaa, T. (1901): Cell Tissue Kinet., 14:9-20. Vilpo, J.A., Kiviniemi, K., and Rytömaa, T. (19-*3): Eur. J. Cancer, 9:515-524. VI SERVOMAA K., RYTÖMAA T. Suicidal death of rat chloroleukaemia cells by activation of the long interspersed repetitive DNA element (L1Rn). Cell Tissue Kinet 1988; 21:33-43. Cell Tissue Kinet. (1988), 21. 33 43. Suicidal death of rat chloroleukaemia cells by activation of the long interspersed repetitive DNA element (LIRr) K. Servomaa and T. Rytömaa Laboratory of Radiobiology, Finnish Centre for Radiation and Nuclear Safety, Helsinki, Finland (Received 4 January 1988; revision accepted 30 March 1988) Abstract. Rat chloroleukaemia cells, maintained in sjspension culture in different media, show rapid exponential growth without cell loss. At about half of the maximal population density the long interspersed repetitive DNA element (LIRn) is suddenly transcriptionally activated without any obvious exogenous reason. Population growth is then inhibited and, within about 24 h after reaching the maximal density, the population undergoes programmed death (apoptosis). Suicidal cell death is caused by sudden incorporation, apparently by retroposition via an RNA intermediate, of about 300,000 copies of the LIRn element into random locations in the cell genome, thus creating lethal mutations. The preceding growth inhibition is associated with repression, to an undetectable level, ofc-Ki-ras expression. Up to the point of massive LIRn incorporation and cell death, all phenomena are quickly reversible by subculturing; medium change alone is not sufficient. Biological implications of these surprising findings are discussed. The long interspersed repetitive DNA element (LI, LINE or retroposon) makes up a considerable proportion of eukaryotic cell genome; in some species this element (6-7 kilobases (kB) in size) consists of as much as 10% of the total DNA (Singer, 1982; Rogers, 1985) and there are, e.g. in rat, about 40,000 copies of the element per diploid cell (d'Ambrosio & Furano, 1987), The functions of the LI element, if any, are unclear at present (Skowronski & Singer, 1985), but it nevertheless seems that this element has been moving within the genome via an RNA intermediate (Snkaki, 1987). The long open reading frames of the LI element encode a putative fusion protein with significant homology to reverse transcriptases (Loeb et al., 1986; Hattori et ai, 1986) and therefore the element appears to drive its own movement. We have observed before (Rytömaa & Servomaa, 1986) that when rat chloroleukaemia cells reach a critical population density in a suspension culture, the LIRn{Rn = Rattus norvegicus) is suddenly activated and cytoplasmic transcripts homologous to this element increase 10-fold. Subsequently, the growth rate of the population is inhibited and, if the inhibition is not released by subculturing, all cells die within about 2 days (Rytömaa & Servomaa, 1986). In this paper we show that programmed cell death (or apoptosis) is caused by sudden massive incorporation, or retroposition, of about 300,000 LIRn copies into apparently random (non-targeted) locations in the cell genome which, in turn, is bound to create lethal mutations in the cells. This programmed death seems to be tightly coupled with the preceding, reversible growth inhibition accompanied with full transcriptional inactivation of the c-Ki-ras oncogene. Correspondence: Prof. T. Rytömaa, Laboratory of Radiobiology, Finnish Centre for Radiation and Nuclear Safety, PO Box 268, SF-O0I01 Helsinki, Finland. 33 34 K. Servomaa and T. Rytömaa MATERIALS AND METHODS Cells and culture Rat chloroleukaemia. induced by gastric instillation of methylcholanthrene (Shay, Gruenstein &. Harris, 1955), was obtained for our laboratory from Dr W. C. Moloney, Boston, in 1969. Since then the cells have been maintained by subcutaneous and intraperitoneal transplantations into young rats (Rytömaa & Kiviniemi, 1970) and, during the last 10 years, by suspension culture in vitro. The cells are grown in different types of culture media, commonly in Dulbecco's modified Eagle medium (DM EM), supplemented with 1% fetal calf serum, penicillin (100 units/ml) and streptomycin (100/ig/ml). The cells can also be grown in a chemically defined medium, DMEM, supplemented with insulin and transferrin, 5-10/ig/ml each. A detailed study of the population kinetics has been published before (Toivonen et ai., 1981); it shows, in particular, that during the exponential growth phase essentially no cell loss occurs. We have recently confirmed this by time-lapse cinematography of cells grown in the chemically defined medium; in this medium the cells attach themselves loosely to the bottom of the culture vessel, and single cells and their progenies are therefore readily amenable to long-term follow-up (unpublished results). All cell counts given in the present paper were made in the Coulter Counter, Model ZM (Coulter). DNA and RNA analyses For isolation of high molecular weight DNA, approximately 10s cells were lysed with 1 % sodium dodecyl sulphate (SDS) in 01 M NaCI, 2 mM EDTA, 50 mM Tris-HCl, pH 7-5, and the cellular proteins were hydrolysed with 200 /ig/ml proteinase K for 1 hr at 37°C. The solution was extracted twice with phenol, and twice with an equal volume of chloroform-isoamylalcohol (24:1). Nucleic acids were precipitated with 2-5 volumes of ethanol, washed in 70% ethanol and dried under vacuum (Hetosicc, Heto). The nucleic acids were redissolved in 1 mM EDTA, 10 mM Tris-HCl (TE), pH 80, and RNA was hydrolysed with 100 /ig/ml pancreatic ribonuclease A (Sigma) at 37°C for 1 hr. Treatment with proteinase K, extractions with phenol and chloroform, and ethanol precipitation were performed as above. DNA was dissolved in T£ and stored at 4°C. Total cytoplasmic RNA was isolated from chloroma cells at different growth phases by lysis in cold buffer (014 M NaCI, 1-5 mM MgC!2,10 mM Tris-HCl, pH 8-6) with 0-5% Nonidet P-40 (BDH), followed by pelleting nuclei, phenol extraction, and ethanol precipitation of RNA (Maniatis, Fritsch & Sambrook, 1982). RNA slot blot analyses (Minifold II, Schleicher & Shuell) were performed with the isolated RNA in a dilution series (40, 20, 10 fi$). RNA blot analysis (Northern blot) for determination of the transcript sizes was performed after electrophoresis of total cytoplasmic RNA (40 /*g). RNA was dissolved in the sample buffer containing 20 mM MOPS (3-[N-morpholino]propane sulphonic acid, Sigma), pH 70, 1 mM EDTA. 40% (v/v) formamide and 2-2 M formaldehyde. Samples were denatured by heating for 10 min at 70 C and electrophoresed in 1% agarose gels (buffer was 50 mM MOPS, pH 70, 1 mM EDTA). The RNA was transferred to nitrocellulose filters by blotting in 20 x SSC (1 x SSC = 015 M NaCl/0015 M sodium citrate). DNA and RNA concentrations were measured spectrophotometrically (Ultrospec 4050, LKB Wallac). Cloning of LIRn from chioroleukaemia Isolated chloroma DNA was digested with the restriction endonuclease BamHI (Boehringer- Mannheim) and fractionated by electrophoresis through 1% agarose gel. A single 5-4 kB band, Suicidal death in chloroleukaemia 35 clearly visible to the naked eye after ethidium bromide staining, was then cut out, electroeluted and dissolved in TE. These DNA fragments were ligated into BamHI-digested pBR322 and introduced into Escherichia coli HB101. Subsequent bacterial culturing and selection of Amp+Ter colonies were performed according to standard procedures (Maniatis et al., 1982). Recombinants hybridizing (under conditions of high stringency, see below) with an isolated 3-5 kB Hindlll band of LI from chloroma DNA were selected and rescreened with an isolated 1-35 kB EcoRI band of chloroma LI (Rytömaa & Servomaa, 1986). On the basis of these screenings three clones pCB4,8 and 16, were selected for the purposes of the present study. For each clone the identity with LIRn was checked by restriction mapping (compared with maps available in the literature, see e.g. Furanoif a/., 1986), and by hybridization with the 1-35 kB EcoRI band of LIMd (3T3 cells), distinct in mouse DNA (cf. Loeb et al., 1986). Final identification with LIRn was obtained by sequencing fragments of the selected pCB clones (results not shown). Hybridizations DNA and RNA filters were baked for 2 hr at 80°C, prehybridized, and then hybridized for 24 hr at 42°C. Hybridizations (and prehybridizations) were performed in Denhardt's mixture (002% each of bovine serum albumin, Ficoll 400 and polyvinylpyrrolidone), 3 x SSC, 50% formamide, 50 mM HEPES, pH 7-2, and 150 j*g/ml salmon sperm DNA. Probes were nick-translated using the kitobtained from Amersham(32P-dCTP, specific activity - 111 TBq/mmol). Approximately 107 cpm were applied to each filter in 20 ml of the hybridization mixture. After hybridizations, filters were washed with two changes in 2 x SSC/0-5% SDS at 24°C for 10 min and two changes in 0 1 x SSC/01% SDS at 55°C for 1 hr, dried, and then exposed to X-ray film (Kodak X-Omat AR using intensifying screens). Autoradiograms were developed according to an automatic standard technique (Sakura QX). Probes Fragments of LIRn cloned from chloroma cells (BamHI inserts in pCB4,8 or 16, or their EcoRI fragments as indicated) were used throughout this study. Judging from partial sequencing (results not shown), the homology of these segments with the normal rat LI element (see d'Ambrosioe/a/., 1986) is >95%; with the mouse sequence (see Loeb etal., 1986) the homology is -85%. The C'Ki-ras probe used in this study is a 0-64 kB FcoRI/Hindlll fragment of the human oncogene, obtained from Dr K. Alitalo (Department of Virology, University of Helsinki). Estimation of LIRn copy numbers DNA was extracted from exponentially growing chloroma cells and from inhibited cultures shortly before cell death, and digested with BamHI. Serial 1:2 dilution of DNA (beginning with 500 ng) was blotted on nitrocellulose filters either alone or mixed with different quantities of cloned LIRn (1 to 4-fold amounts of 150, 75 or 375 ng of BamHI-digested pCB4). The samples were then hybridized with a nick-translated probe (BamHI-digested pCB4). Signal intensities were measured from autoradiograms by scanning with a densitometer (Laser-Microdensito- meter MDM-1, Donor), and also by counting 12P activities of each dot by a liquid scintillation counter (LKB Wallac, model 1215 Rackbeta) LIRn copy numbers were then calculated by noting that 100 ng of LIRn (pCB4) DNA is equal to 9-4 x 10'' copies, ?nd assuming that a hyperdiploid chloroma cell contains an average of 10 pg of DNA. 36 K. Servomaa and T. Rytömaa RESULTS Growth aid morphology of the cells The growth curve of chtoroieukaemia cells (usually maintained in 1% fetal calf serum) is shown schematically in Fig. 1. Some of the sampling times for RNA extraction are shown by arrows, and the critical population density (= activation of the LIRn element) is also indicated (see below). This point is reached at approximately half of the maximal population density, a little less than one cell cycle time before the cessation of net growth. The maximal population density and also the critical density are different in different culture media, but the variation is not very large (details not shown). The highest values are reached in 10% fetal calf serum (over 2 x 106 cells/ml) and the lowest in the chemically defined medium (about 0-7 x 106 cells/ml). Typical examples of cell morphology at an early stage of the population death are also shown in Fig. 1. Light microscopic analysis of dying cells suggested to us that the cells enter a programmed death (apoptosis; Wyllie, Kerr & Currie, 1980; Beaulaton & Lockshin, 1982); hi 0 10 20 30 40 50 60 70 80 90 100 Culture time (hr) Fig. I. (a) Growth curve of chloroleukaemia cells propagated in suspension culture in vitro. Actual data points are not given as the growth rate is different in different media (for numerical data, see Toivonen et ai, 1981; Rytömaa k Servomaa, 1986; Fig. 2), (b) Dying chloroina cells in cytocenlrifuge preparations, sampled at times shown in the growth curve. Note that the dying cells display unequivocal nuclear changes of apoptosis, and that a few normal* looking mitoses are present adjacent to these cells. Giemsa staining. * 2400. Suicidal death in chloroleukaemia 37 particular, cell morphology allows necrosis to be excluded. Note also that at this stage some mitoses are still seen adjacen» (actually present in loose cell clumps in culture) to dying cells (Fig. 1). According to light microscopy and failure of subculture, and also to time-lapse cinematography (results not shown), essentially all cells appear dead by about 2 days after the cessation of net growth. We feel that all cells die by apoptosis but cannot exclude the possibility that the last cells suffer from toxic necrosis. Transcriptional activation of LIRn Transcriptional activation of the LIRn element at the critical population density is shown in Fig. 2. Note that the time difference between population sizes 41 x 10s and 51 x 10s cells/ml (Fig. 2) is 4 hr for cells with a doubling time of 13 hr (Fig. 1) and hence the observed 10-fold increase in the LIRn transcript level is sudden. This increase is similarly detected with a probe originating from the 5' half of the LIRn element (EcoA, a 2-3 kB EcoRI fragment isolated frompCB4; Fig. 2) and with a probe consisting of a 3' segment of the element (EcoB, a 1 35 kB EcoRI fragment isolated from pCB16, see below, Fig. 4). These findings suggest that either a single full-length transcript or several transcripts representing different parts of the LIRn element are present. Northern blot analysis (Fig. 3) showed that the latter suggestion appears to be true, and that LIRn homologous transcripts vary in length from about 10* to a few hundred nucleotides. Note that 10 kB transcripts are larger than the full-length LIRn and these transcripts are therefore possible hybrid (read-through) molecules of unique sequences. Screening of a cDNA library prepared in XgilO phage from cytoplasmic poly(A)+-selected chloroma RNA, isolated after the activation of the LI element, showed that about 0-3% of the clones hybridize strongly with L/-specific probes (details not given). This result indicates that LI transcripts must be one of the most abundant mRN A species in grt wth-inhibited chloroma cells. Furthermore, because a large fraction of the positive clones hybridize with both 5' and 3' segments of the LI, the apparent lull-length transcripts are not rare. LIRn transcript levels remain high or even increase further at higher population densities (see below, Fig. 4), but they quickly return by subcuituring to the baseline level typical of the exponential growth phase. This conclusion is obvious from the comparison of Figs 1 (growth curve) and 2 when one notes that subcultures are routinely made from cultures at or near the maximal cell density, i.e. well after the sudden (and sustained; see below, Fig. 4) increase in the LI transcript level. If the subcuituring is delayed (24 hr or so after reaching the maximal density) the LIRn transcript level does not decrease. However, the moment of entering this stage of irreversibility has not been well defined. 40 20 10 30 350 410 5,0 4tfP Fig. 2. Transcriptional t;tivation of LiRn in chioroma cells. Numbers on the left indicate population sizes (cells xml"1 x 1(H), c/. arrows in Fig. 1. Dot blot analysis of total cytoplasmic RNA (/jggiven at top of figure) probed with a 5' segment of cloned LIRn (EcoA fragment from pCB4). 38 K. Servomaa and T. Rytömaa 5 tO 410 350 30 i 28S« 18S" F|t> 3. Nortbern blot of cytoplasmic RNA isolated at different stages ofchloroma cell growth (numbers at top of figure give cells x ml'1 x 10"1: cf. Figs 1 and 2). A 3' segment of cloned LlRn (EcoB fragment from pC£/6) was used as the probe. Positions o.- 28S and 18S rRNA bands are indicated as size markers. Proto :Eo»A Proto: E 40 20 10 40 20 10 210 2lt •II 111 •» — m it 7M 9 «9 Proto: e-KI-ro» 40 20 10 211 111 7M Fig. 4. Expression of c-Ki-ras and LlRn in different phases of chloroma cell growth (numbers give cells x ml-1 x 10-J; cf. Figs 1, 2 and 3). Dot blot analysis of total cytoplasmic RNA 0*g given at top of figure), probed with human c-Ki-ras and LlRn (5' and 3' EcoRI fragments of LlRn, isolated from pCB4), Medium change alone at or near the maximal population density allows some further (slow) growth but does not rescue the population if the population density is not diluted (results not shown). This suggests that maintained expression of the LlRn element is associated with the cell population density as is triggering of the LI activation. These associations were not studied further in the present study. Suicidal death in chioroieukaemia 39 Expression of c-Ki-ras It was expected that c-Ki-ras oncogene would be activated in the methylcholanthrene-induced chioroieukaemia. This expectation was based on the reported common involvement of c-Ki-ras in methylcholanthrene-induced tumours (Eva & Aaronson, 1983) and on our own finding that chloroma DNA transforms mouse ST3 cells at a high frequency in the conventional transfection assay (results not shown). We therefore studied Kirsten-ras expression in association with the activation of the LIRn element. The results (Fig. 4) show that LIRn and c-Ki-ras behave in the opposite way: Kirsten-ras is actively expressed at the exponential growth phase but is repressed to an undetectable level when growth is inhibited and LIRn transcript level is high. Note also that these results (Fig. 4) serve as mutual controls, indicating that differences in the signal intensities are not caused by variable amounts of RNA present in the dot blots. Amplification of LIRn in the genome The observed transcriptional activation of LIRn and the subsequent sudden death of chioroieukaemia cells in dense cultures without apparent exogenous reason (medium change fails to rescue the population) suggested to us that the explanation might be found in the presumed retroposon nature of the LI element. Although it is clear that retroposition of the LI element is not a common contemporary phenomenon in animal ceils (as opposed, e.g. to the movements of transposons in yeast; see Boeke et al., 1985), we reasoned that it might nevertheless occur in the exceptional case of chloroma cell death. Dot blot analysis of chloroma DNA indicated that, compared with cells in the exponential growth phase (Fig. 5), a huge excess of LIRn elements becomes detectable in the cell genome just before the morphological death. The actual mechanism of the massive amplification, leading to about a 7-fold increase, from about 45,000 to about 340,000 copies per cell genome (see Figs 5 and 6 and Materials and Methods), is not obvious from the results shown here. However, the present finding that LIRn transcript level first increases 10-fold in the cytoplasm (Figs 2, 3 and 4) — and that the element may encode a peptide homologous to reverse transcriptases (Hatton et al., 1986) — suggests that the amplification has occurred via an RNA intermediate. So far, however, we have failed to detect unequivocally reverse transcriptase activity in chloroma cells, perhaps because the enzyme is active only for a very short period before the cell death. Nevertheless, sudden incorporation of about 300,000 LIRn copies or its fragments (Figs 5 and 6) into apparently random (see, however, Furano et al., 1986), non-targeted locations in DNA is bound to destroy the genome and hence to lead, by lethal mutations, to cell death. Growth kinetics (sec Fig. I) and preliminary findings in time-lapse cinematography (results not shown) suggest that there is a buiid-up of cells in the G, phase of the cell cycle, followed by apoptosis. Light microscopic findings(c/ . photomicrographs in Fig. 1) indicate that cell death does not occur by failure of mitosis. 500 250 125 Growing ^_»* ^_w ^ cells ^nn» ^nn» ^*- Dylng ^^^^k ^•JB^ ^m^^ calls f^^V ^P^ ^|F Fig. 5. Amplification of LIRn in chloroma cell genome just before population death. Growing cells refer to the exponential growth phase and dying cells to the phase indicated in Fig. I. Dot blot analysis of BamHI-digesled DNA. probed with cloned LIRn (pCB4). Amounts of DNA blotted are given at top of figure (ng). 40 K. Servomaa and T. Rytömaa 500 250 f25 Chloroino DNA -» l50/75/;7 5 Mixed 300/150/75 with pCB4 450/225/112 5 600/300/150 Fig. 6. Estimation of copy numbers of LIRn elements in chloroma cell genome after death-associated amplification (ng of DNA shown at top of figure). The lower tour rows contain mixtures of amplified DNA and known amounts of cloned LIRn (ng olpCB4 shown on the right;se e Materials and Methods). Dot blot analysis of BamHI-digested DNA, probed with pCB4. Fig. 7. Massive cell death in a subcutaneous chloroma tumour, following prolonged intraperitoneal injections of partially purified granulocyte extracts (for details, see Rytömaa k Kiviniemi, 1969, 1970). Re-evaluation of the sudden regression of the growth-arrested tumours suggests that this massive in vivo cell death was also caused by activation of the LIRn. The mechanism which triggers transcriptional LIRn activation and subsequent amplifica tion in chloroma cell DNA is not indicated by the present results, apart from the finding that both phenomena are normally associated with dense populations We have, however, also observed that occasionally chloroma cells begin to decelerate growth rate even before they have reached the usual critical population density and that it is then difficult to subculture the cells from a normally safe density. The reason for an occasional, apparently premature activation of the LIRn element remains unclear at present. Growth inhibition and ceil death in vivo Inhibition of growth seems to be the most consistent finding preceding chloroma cell death in vitro, We have observed this, or at least a macroscopically identical sequence of events, even in in vivo conditions (Fig. 7), when rats bearing subcutaneous chloroma tumours were treated with a Suicidal death in chioroleukaemia 41 chemically undefined inhibitory substance called granulocytic chalone (see also Rytömaa & Kiviniemi, I969). In both cases, in vivo as well as in vitro, the similar massive cell death is almost instantaneous and involves cells which, even by usual morphological criteria, do not seem to suffer from a cytotoxic attack (see Fig. 1). We believed earlier that immunological mechanisms might be responsible for the in vivo death of inhibited chloroma cells (Rytömaa & Kiviniemi, 1970), but of course these mechanisms do not operate in vitro. Simple toxicity of the in vivo injected leucocyte extract was also considered, but excluded on different grounds (Rytömaa & Kiviniemi, 1969, 1970); in the present in vitro culture no exogenous material was used. Both in vivo and in vitro, cell death is thus preceded by growth inhibition, although the spontaneous in vitro mechanism may not necessarily involve 'granulocytic chalone'. DISCUSSION In this paper we have described a surprising phenomenon where immortal, frankly malignant leukaemia cells commit apparent group suicide by self-induced lethal mutations. This process involves sudden massive incorporation, probably by retroposition through an RNA intermediate, of about 300,000 copies of the long interspersed repetitive DNA element into the cell genome. It appears reasonable to assume that the element is incorporated into random locations — if the massive uptake were targeted, then the process would be even more peculiar, at least from a philosophical point of view. One of the obvious questions arising from the present findings is the triggering of the observed series of events. The onset of the process seems to be spontaneous with no connection to any deliberately induced variable except population density. Programmed cell death (Wyllie etal., 1980; Beav.laton & Lockshin, 1982) is a descriptive answer but, of course, this answer does not suggest anything about the activation of the death programme. Light microscopic findings show that the cell death does have the features of apoptosis although biochemical markers, such as activation of endonucleases (Wyllie, Duvall & Blow, 1984) have not been sought. Furthermore, it is not clear from the present study whether the execution of the programme in chloroma cells is similar to processes leading to a biologically significant, controlled cell death (apoptosis as opposed to necrosis). It is nevertheless tempting to speculate, even in the absence of any direct evidence, that normal cells may indeed be occasionally killed by similar self- induced lethal mutations. If not, then the acquisition of this mechanism into rat chloroma cells through some sort of clonal evolution would be a biological oddity, especially because chloroma cells are immortal and are obviously not evolving toward programmed death. Some findings seem to support the idea that retroposition of DNA elements may actually be an important ancient mechanism in programmed cell death. Thus coph in Drosophila and Ty in yeast, which are movable genetic elements and resemble the LI family of higher eukaryotes, are transcriptionally activated by environmental stresses such as elevated temperature and exposure to certain chemicals or u.v. irradiation (Strand & McDonald, 1985; Rolfe, Spanos & Banks, 1986). Furthermore, Boeke et al. (1985) have established that increased levels of Ty transcripts are indeed correlated with increased transposition of Ty elements, and that the transposition proceeds via an RNA intermediate. These experiments also indicated that transposition events were accompanied by growth retardation in the yeast and that the process may well contribute to the induction or mutations and other chromosomal aberrations (Boeke et al., 1985). At the critical chloroma cell density, approximately one ceil cycle away from the final size, the LI element is suddenly activated at the transcriptional level. This activation, which is 42 K. Servomaa and T. Rytömaa immediately followed by strong growth inhibition, is reversible by subculturing, and the reversibility is maintained past the stage where population net growth has fully ceased. Interestingly, the inhibition — and the subsequent population death — cannot be prevented by changing the culture medium only; dilution of the population back to a tow density appears obligatory. On the other hand, the onset of the phenomenon is medium-dependent at least to some extent, because the critical population size and also the cell death phase are reached at different densities in different cultui" media. Furthermore, occasionally growth inhibition begins prematurely; subculturing is then difficult although the population density is still 'safe'. The apparent coupling of growth inhibition, both in vitro and in vivo, and programmed cell death in chloroma cells is intriguing. It may not seem totally unreasonable to think that a miniscaled, possibly targeted, retroposition of the Ll element into the cell genome would first lead to growth inhibition and that cell death only follows when the mechanism results in some sort of explosive integration. However, the full and quick reversibility of the inhibition by subculturing would seem to require excision of the integrated Ll element from its new location. If not excised, then one would expect accumulation of inhibitory mutations in chloroma cells and subsequent slow deterioration of population growth; this is, however, exactly the opposite to what we have seen during the many years of chloroma cell maintenance (regarding population kinetics, see e.g. Vilpo & Rytömaa, 1973; Toivonen et ai., 1981, and the present situation). Repeated and targeted integration and excision of the Ll element would also seem necessary on other grounds: active expression of the c-Ki-ras oncogene, which is almost certainly correlated with the malignant character of chloroma cell growth, is fully repressed when Ll is transcriptionally activated, but is again derepressed when the cells are subcultured (and LI is deactivated) (see Fig. 4). We believe, however, that regulated jumping of the Ll element in chloroma genome is highly unlikely and therefore that the growth inhibition, as opposed to cell death, is not based on the transposon-like behaviour of this element. Nevertheless, it is impossible to ignore the observed close association between transcriptional activation of the Ll element and growth inhibition, inverse behaviour of c-Ki-ras and Ll expressions, and quick reversion of all these phenomena by subculturing. Further results of the intriguing behaviour of the Ll element in chloroma cell inhibition will be reported in another paper. For instance, a possible relationship between the LIRn transcripts and the high abundance antiproliferative mRNA, detected in senescent human diploid fibroblasts (Lumpkin et ai, 1986), needs to be clarified. REFERENCES BEAULATON, J. & LoCKsHIN, R. A. (1982) The relation or programmed cell death to development and reproduction: comparative studies and an attempt at classification. Int. Rev. Cytol. 79, 215. BOEKE, J.D., GARFINKEL, D.J., STYLES, C.A, & FINK, G.R. (1985) Ty elements transpose through an RNA intermediate. Cell 40, 491. D'AMBROSIO, E. & FURANO, A V. (1987) DNA synthesis arrest sites at the right terminus of rat !ong interspersed repeated (LINE or LIRn) DNA family members. Nucleic Acids Res. 15, 3155. D'AMBROSJO, E., WAITZKIN, S.D., WITNEY, F.R., SALEMME, A. & FURANO, A.V. (1986) Structure of the highly repeated, long interspersed DNA family (LINE or LIRn) of the rat. Mol. Cell. Biol. 6, 411. EVA, A. & AARONSON, S.A. (1983) Frequent activation of c-kis as a transforming gene in fibrosarcomas induced by methylcholanthrene. Science 220, 955. FURANO, A.V,,SoMERViu.EtC,C.,TsiCHLls, P.N. AD'AMBROSIO, E. (1986) Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random Nucleic Acids Res 14, 3717. HATTOÄJ, M., KUHARA, S.. TAKBNAKA, 0. & SAKAJCJ, Y. (1986) Ll family of repetitive DNA sequences in primates may be derived from a sequence encoding a reverse transcriptase related protein. Nature 321, 625. Suicidal death in chioroleukaemia 43 LOEB, D.D., PADGETT, R.W.. HARDIES, S.C., SHEHIE, W.R., COMER, MB. EDGELL, M H & HUTCHISON. C A (1986) The sequence of a large L1 Md element reveals a tandemly repeated 5' end and several features found in retrotransposons. Mol Cell. Biol. 6, 168. LUMPKIN, C.K. JR. MCCLUNG, J.K., PEREIRA-SMITH, OM. & SMITH, J.R. (1986) Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts. Science 232, 393. MANIATIS, T , FRITSCH, E.F. & SAMBROOK, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, New York. ROGERS, J.H. (1985) The origin and evolution of retroposons. Int. Rev. Cytol. 93, 187. ROLFE, M., SPANOS, A. & BANKS, G. (1986) Induction of yeast Ty element transcription by ultraviolet light. Nature 319, 339. RYTÖMAA, T. & KIVINIEMI, K. (1969) Chloroma regression induced by the granulocytic chalone. Nature 222,995. RYTÖMAA, T. & KIVINIEMI, K. (1970) Regression of generalized leukaemia in rat induced by the granulocytic chalone. Eur. J. Cancer 6, 401. RYTÖMAA, T. & SERVOMA/, K. (I986) Identification of a putative growth inhibitor gene in rat chioroleukaemia ceils. In: Biological Regulation ofCell Proliferation (Serono Symposia Publications, Raven Press, vol. 34) (Ed. by Baserga. R, Foa, P., Metcalf. D. & Polli, E.E.), p. 93. Raven Press, New York. SAKAKI. Y. (1987) RNA-mediated (?) dispersion of the LI family of long interspersed repetitive DNA in mammalian genomes. Mol. Biol. Med. 4, 193. SHAY, H . GRUENSTEIN, M. & HARRIS, CH. (1955) Experimental leukemias in the rat. Acta Haemal. 14, 337. SINGER, M.F (1982) Highly repeated sequences in mammalian genomes. Int. Rev. Cytol. 76, 67. SKOWRONSKI, J. & SINGER, M.F. (1985) Expression of a cytoplasmic LINE-1 transcript is regulated in a human teratocarcinoma celt line. Proc. Natl. Acad. Sci. USA 82, 6050. STRAND, D.J. & MCDONALD, J.F. (1985) Copia is transcriptionally responsive to environmental stress. Nucleic Acids Res 13,4401. TOIVONEN, H , FOA, P., PAILE. VV. & RYTÖMAA, T. (1981) Detailed kinetic analysis of Shay chioroleukaemia cell population propagated in permanent suspension culture in vitro. Cell Tissue Kinet. 14, 9. VILPO, J.A. & RYTÖMAA, T. (1973) Proliferation kinetics of Shay chioroleukaemia cells grown in diffusion chambers in vivo. Cell Tissue Kinet. 6, 489. WYLLIE, AH, KERR, JFR 4 CURRIE, A.R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251. WYLLIE, AH, DUVALL, E. & BLOW, J.J. (1984) Intracellular mechanisms in cell death in normal and pathological tissues, in: Cell Ageing and Cell Death (Seminar Series of Society for Experimental Biology, vol. 25) (Ed. by Davies, I. & Sigee, D.C), p. 269. Cambridge University Press, Cambridge. VII SERVOMAA K., RYTÖMAA T. UV light and ionizing radiations cause programmed death of rat chloroleukaemia cells by inducing retropositions of a mobile DNA element (L1Rn). Int. J. Rad. Siol. 1990; 57: 331-343. INT. J. RADIAT. BIOL., 1990, VOL. 57, NO. 2, 331-343 UV light and ionizing radiations cause programmed death of rat chloroleukaemia cells by inducing retropositions of a mobile DNA element (LIRn) K. SERVOMAA and T. RYTÖMAA Laboratory of Radiobiology, Finnish Centre for Radiation and Nuclear Safety, P.O. Box 268, SF-00101 Helsinki, Finland f Received 3 July 1989; revision received 15 September 1989, accepted 22 September 1989) The long interspersed repetitive DNA, LI or LINE, is a class of mobile genetic elements which can amplify in the cell genome by retroposition, i.e. by a mechanism similar to that of retroviruses. We have shown before that in rat chloroleukaemia cells, maintained in suspension culture in vitro, this element is spontaneously transcriptionally activated at about half of the maximal popul ation density. About 24 h later an explosive amplification of the LI element is seen in DNA: about 300,000 copies are inserted into apparently random locations in the cell genome, thus creating an outburst of lethal mutations. Dead cells display morphological features typical to programmed death. The present results show that UV light and ionizing radiation induce rapid, premature activation of the LIRn element during the fast exponential growth of chloro leukaemia cells, and that also this exogenously induced activation is followed by programmed cell death. Transcriptional activation of the LIRn element can be very strong after the UV exposure: at least 70-fold. Severe hyperthermia, lethal to the cells, does not lead to LIRn activation (actually a marked suppression is seen) and the mode of phenomic death is necrosis. Some biological implications of the results are discussed. 1. Introduction LI or LINE family, the long interspersed repetitive DNA element, is a class of highly repeated DNA sequences present in all mammals studied to date (Loeb et al. 1986, D'Ambrosio et al. 1986). This mobile genetic element, which resembles some other transposable elements present in different organisms, seems to amplify by integration of DNA copies of RNA transcripts into the cell genome (retroposition, i.e. by a mechanism similar to that of retroviruses). The two open-reading frames of LI encode a putative fusion protein with significant homology to different retro virus proteins including, in particular, reverse transcriptase (Hattori et al. 1986, Fanning and Singer 1987). Many mobile genetic elements, e.g. some of those present in yeast and Drosophila, are known to be transcriptionally activated by environmental stresses such as elevated temperature, exposure to certain chemicals and UV irradiation (Strand and McDonald 1985, Rolfe el al. 1986, Morawetz 1987), To the best of our knowledge, however, similar activation has not yet been reported for the mammalian LI element (see also UNSCEAR 1988). Another line of evidence important in the context of the present study involves apoptosis (Wyllie et al. 1980, Beauiaton and Lockshin 1982, Kerr et al. 1987). This form of cell death is believed to be an active, intrinsically programmed pheno menon which has been suggested to occur in controlled, selective and biologically 0020 76KVW S.VCK» n I'Of) Taylor & trancis l.ltf 332 K. Servomaa and T. Rytömaa meaningful elimination of cells. The onset of apoptosis is triggered by factors that may be intrinsic as well as extrinsic to the cell; among the latter are UV light (Weedon et al. 1979, Gunn et at. 1983) and ionizing radiation (Potten 1977, Hendry et al. 1982, Yamada and Ohyama 1988). Intrinsic factors are probably involved in many cases where programmed death appears to be spontaneous; these cases include normal embryonic and foetal development, cell turnover in many normal adult tissues and, apparently as an inherent characteristic, neoplastic growth (Collins et al. I989). We have recently made the surprising finding, which associates mobile genetic elements with proprammed cell death, that the long interspersed repetitive DNA element (LI or LINE) is spontaneously activated in rat chloroleukaemia cells in vitro and apparently also in vivo, and that this leads to cell death through self- induced lethal mutations (Servomaa and Rytömaa 1988). Dead cells display nuclear changes typical to apoptosis. Expression of the LIRn element (Rn — Rattus norveg- icus) occurs suddenly at a critical population density and, if the process is not reversed by subculturing, the transcriptional activation is followed by an explosive amplification of LIRn in the cell genome: about 300,000 copies of the element are incorporated into random locations (Servomaa and Rytömaa 1988). The onset of the self-destructive process is spontaneous (but associated with population density) with no obvious connection to mechanisms such as accumulation of toxic meta bolites in the culture medium; for instance, medium change alone does not save the population. We now show that exposure of chloroleukaemia cells to UV light causes rapid and premature activation of the LIRn element, and that this ieads to similar ceil death as the spontaneous LIRn activation. 2. Materials and methods 2.1. Cell cultures Rat chloroleukaemia cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 per cent foetal calf serum, penicillin (100 units/ml) and streptomycin (100/jg/ml). The cultures were incubated at 37°C in humidified air containing 5 per cent C02. 2.2. Growth curves Chloroma cells were seeded in 30-mm tissue culture dishes (Sterilin) at an initial concentration of 2-3 x 104 cells/ml in 2 m! of the medium. All cell counts were made in the Coulter Counter, Model ZM (Coulter). Two independent cultures were sampled three times a day for 5 days, and each culture was sampled only once. 2.3. UV irradiation The cultures were irradiated on a commercial UV-light table (Fotodyne Inc.) routinely used for visualization of nucleic acids, e.g. in electrophoresis gels. Spectral irradiance of this UV source is shown in figure 1 (actual range measured was from 200 to 400nm, but irradiance below 250nm was essentially zero); dose rates at the table surface are given in table 1. UV irradiation with a fairly wide wavelength-action spectrum was used in the present study, mainly because we could not decide in advance which type of UV- induced damage, if any, would be most effective in activating LIRn expression in chloroma cells, f n particular, we did not want to confine ourselves to UV irradiation at 254nm, a wavelength in the sunlight which does not reach the surface of the Radiation, mobile DNA and programmed death 333 •10-» 3 2.5 1 2 cs I 0.5 240 MO 280 300 320 340 360 3S0 400 Wavelength (am) Figure 1. Spectral irradiance from the commercial UV-Hght table {Fotodyne Inc.) used in the present study. Irradiance was measured (Optrovic Spectroradiomerer, Model 742) at a distance of 35 cm from the table surface, at T= 24°C. earth, but wanted to include wavelengths from 290 to 320 nm, which are believed to be the most important with respect to the carcinogenic potential of sunlight (see Setlow 1974). Because we did not apply defined-wavelength UV radiation, we made an approximate quantitative comparison between the UV source used and ionizing radiation in inducing single- and double-strand breaks in naked DNA. To this end (details not given), a supercoiled recombinant plasmid (pCB4, about 9-8 kb in length; see Servomaa and Rytömaa 1988) was irradiated in an agarose gel, and the number of nicked molecules was estimated by comparing intensities of DNA bands of supercoiled (= intact) and nicked molecules (figure 2). According to this analysis, exposure for 2 min from the present source induced strand brea! " in about half of the plasmid molecules; a similar effect was obtained with about 25 Cry of ionizing radiation (either /?-rays from a 90Sr source or the X-rays used in the present study). 2.4. X-irradiation The cultures were irradiated with X-ray• using a Valmet BR 1001 three-phase generator (W anode X-ray tube, 110 kV tube voltage, 1 -7 mm Al filtration, dose rate 0-2 Gy/min). The total dose to the culture was 3 Gy. Table 1. Measured LIV irradiances at the Fotodyne table surface. Exposure times of the cultures were 2,10 and 30s, as indicated in the text. Wavelength . Irradiance (nm) ' (W/m:) 254 (UVC) 04 28ft-315 (UVB) 60 316-400 (UVA) 15 334 K. Servomaa and T. Rytömaa A B Figure 2. An example of the estimation of single- and double-strand breaks, induced in immobilized naked DNA by UV light. A supercoilcd re combinant plasmid (pCB4, about 9-8 kb; see Servomaa and Rytömaa 1988) was moved by electrophoresis into the position indicated by the upper arrow, and then exposed to UV light. In subsequent electrophoresis, supercoiled mole cules were further moved into the position in dicated by the lower arrow (see lane 8) while nicked molecules, which have a strongly re tarded mobility, remained almost in the initial position (see lane A). Note that the uppermost bands are caused by nicked and linearized plas- mids which were present in the original sample. The exposure used in this example was 3 min (cf. table 1 and figure 1), and more than half of the molecules were nicked (cf. the band intensities shown by the arrows). 2.5. Irradiation with f 3HJthymidine [3H]thymidine (Amersham, specific activity 925 GBq/mmol) was added to the cultures at an early growth period, and the cultures were then incubated at 37°C for 4 h or 24 h, during which time the chloroma cells were chronically exposed to tritium. The concentration of the [3H]thymidine added ranged from 7-4 to 37kBq/ml. 2.6 Hyperthermia For heat treatment, the cultures (fresh medium, cellularity <10s/ml) were incubated at the desired temperature in humidified air containing 5 per cent C02. This model was adopted because we wanted to avoid pH shock to the cells and other possible changes in the culture medium arising from evaporation. Under the treatment conditions the temperature of the medium reached the final level slowly, within 1 h, and we therefore adjusted the treatment periods and temperatures according to the effects on population growth. Cell counts (cultures transferred to 37°C after the heat treatment) showed that heat treatment at 42°C up to 4 h (including the time needed for temperature rise) did not affect the population growth at all, or affected it minimally; after the treatment at 43°C for 3 h the net growth was drastically reduced (Td changed from 11 h to about 50 h). On the basis of the calibration trials, two heat treatments were adopted for the actual experi ments: 43 C for 3h, and 44'C foi 4h. 2.7. RNA isolation and analysis Total RNA (cytoplasmic and nuclear) was isolated by lysing cultures in 4 M guanidinium isothiocyanate, 20 mM sodium acetate (pH 5-2), 0-1 mM diothiothreitol Radiation, mobile DNA and programmed death 335 (DTT), and 0-5 per cent JV-lauryl sarcosine (Sarkosyl). RNA was purified by centrifugation through a cushion of 5-7 M CsCl and 01 M EDTA (pH 7-5). The RNA pellet was then suspended in TE (lOmtu Tris (pH7S), 01 mM EDTA), extracted once with an equal volume of chloroform-1-butanol (4:1), and pre cipitated in ethanol. Cytoplasmic RNA was extracted from cells lysed in cold buffer (0-14 M NaCl, 15 mM MgCl2, 10mw Tris-HCl, pH 8-6) with 0-5 per cent Nonidet P-40, and the nuclei were pelleted. After phenol-chloroform extraction the RNA was precipitated in ethanol. The RNA was then suspended in TE and analysed by direct slot or dot blotting (Minifold I or II) of the RNA onto nitrocellulose (Schleicher and Schuell). The blots were baked for 2 h at 80°C, and then prehybridized for at least 6 h at 42°C in the following buffer: 50 per cent formamide, 1 x Denhardt's (0-02 per cent bovine serum albumin, 0-02 per cent Ficoll 400 and 0-02 per cent polyvinylpyrrolidone), 3 x SSC, 50mM HEPES, pH7-2, containing 150/xg/ml salmon sperm DNA. The blots were then hybridized for 24 h at 42CC in the same mixture with 32P nick- translated probes (specific activity about 108cpm//*g). Washing was done under stringent conditions. Autoradiography was performed by exposure of the X-ray film (Kodak X-Omat AR using intensifying screens) and using standard development (Sakura QX). Signal intensities were measured by scanning the autoradiograms with a 300A Computing Densitometer (Molecular Dynamics). 2.8. Probes Appropriate fragments of LlRn, cloned from chloroleukaemia cells (Rytömaa and Servomaa 1986, Servomaa and Rytömaa 1988), were used throughout this study. The main probe used here is a 5-5-kb BamHI fragment of chloroma LlRn (recombinant plasmid pCB4\ see Servomaa and Rytömaa 1988), covering the two reading frames of the element almost in full. 3. Results 3.1. U V ir radial ion Exposure to UV for 30 s (see figure 1 and table 1) activates the LlRn element in rat chloroleukaemia cells almost instantaneously (figure 3 and table 2): 2 h after the exposure the level of cytoplasmic LlRn transcripts has increased about 10-fold compared with unirradiated controls. The measured signal is, in fact, stronger than that seen in the spontaneous LlRn activation occurring later at the critical population density (figure 3 and table 2; see also Rytömaa anci Servomaa 1986, Servomaa and Rytömaa, 1988). A few hours after the UV exposure, growth of the chloroleukaemia cells ceases altogether; on the following day, about 20 h after the exposure, most of the cells are apoptotic in morphology (figure 4), and the LlRn transcript signal has increased 70-fold (figure 3 and table 2) compared with the baseline level. If the exposure time is shortened to 10 s, the growth pattern of the cell population is similar to that seen after the larger dose. The only actual difference between the two exposures is in the speed of the transcriptional response of the LlRn element: if the dose is reduced an increase in the LlRn transcript level is not yet detectable at 2 h (figure 3 and table 2). On the following day, however, an 336 K. Servomaa and T. Rytömaa 20 10 Unexposed control *^ *** 10S "*" 2 h after uv 30 S Spont. activated LI lOs 24 h after UV 30 S Figure 3. Transcriptional activation of LlRn in chloroleukaemia cells by UV light. Slot blot analysis of total cytoplasmic RNA (fig given at top of figure) probed with cloned LlRn (5-5-kb BamHI fragment from pCB4). Note that in this figure the control signal, obtained with RNA isolated from another chloroma population, is exceptionally high, and has not been used in the actual quantitative comparisons given in table 2. Table 2. Induction of LlRn transcription in rat chloroleukaemia cells by exposure to different extrinsic factors. Relative signal intensity Exposure At 3 h Next day Nonef 1 1 UV hghtj 10s 1 47 30s 11 73 Acute X-rays 3 Gy 0-4 5 8 f3H]Tdr 7 4 kBq/ml 1 2 6 37 kBq/ml 1 3-6 Heat treatment 44°C, 4 h 0-6 0 4 The values give relative signal intensities (unexposed control cells, before reaching the critical population densityf, =1), obtained by densitometric measurements of slot blot autoradtograms of cytoplasmic RNA (see also figures 3 and 6-8). tAt the critical population density (about half of the maximal density for any particular medium; see Servomaa and Rytömaa 1988), spontaneous activation occurs and gives a relative signal intensity of about 5, J For spectral fluences see §2. Most of the total dose given is produced by UVB (280-31S nm; see figure I and table 1). increase of about 50-fold is seen, and there is not much difference in the signal intensity between the two exposure groups (figure 3 and table 2). If the UV dose is further reduced by a factor of 5 (2 s exposure), growth rate of the cell population is detectably inhibited, but many (or most) of the cells survive and net population growth continues. The transcript level of the LlRn element is Radiation, mobile DNA and programmed death 337 A <&l>: 1 . B •mito. * J- • • AW IRK _i_ X^F » • ^^•^•/%-> Figure 4. Death of chloroleukaemia cells induced by U V light (A and B) and by heat treatment (C). After the UV exposure, extensive cell death is seen with characteristic nuclear changes. After the severe hyperthermia, dying cells display morphological changes typical to necrosis. Note also that in the chloroleukaemia population apoptotic mode of cell death can be very extensive (A) and yet scattered mitoses are still seen adjacent to these cells (B); following lethal heat treat ment and subsequent cell necrosis, mitoses are not present (C). Cyto- centrifuge preparations, May- Gninwald-Giemsa staining, magni fication (x 450). slightly increased compared with unirradiated cultures, apparently because the element is activated only in some of the cells. Accordingly, only a fraction of the cells die later (results not shown). 3.2. Exposure to X-rays ' An acute X-ray dose of 3 Gy affects chloroma cell growth rate (fig. 5) somewhat more than the lowest UV dose tested. The kinetics of the transcriptional activation of the LIRn element seem to differ slightly between ionizing and UV radiation exposures. After the X-ray dose, the initial response in LIRn transcription is a clear decrease rather than an increase, and this decrease is maintained at 4 h after the exposure (figure 6 and table 2). On the following day, however, a distinct increase in the LIRn transcript level is seen (figure 6 and table 2), but the activation of the LIRn DNA element is less marked than after the large UV doses (figures 3 and 6, and table 2). It appears that after the acute X-ray dose many cells die from mitotic failure (Scott and Zampetti-Bosscler 1980) as well as apoptisis (intermitotic death). 338 K. Servomaa and T. Rytömaa CELLS/ml Spontaneous activation of LlRn o Unirradiated 10* Activated L1Rn distinct ( after X-rays I 10* X-rays l3Gy) I I I I 1 1 L 0 10 20 30 40 50 60 CULTURE TIME I hours ) Figure 5. Effect of X-rays (3 Gy) on chlorokcukaemia cell growth. Transcriptional activ ation (induced or spontaneous) or the LIRn element is shown in the figure by arrows (in irradiated cells, the actual time of activation is somewhere between 4 and 20 h; see text). Apoptosis begins in control cultures soon after the cessation of net growth; in irradiated cultures it is seen by 24h after the exposure. All cell-count points are from independent cultures. 3.3. DNA irradiation by 13H •thymidine DNA-bound tritium, incorporated into the cells by exposure to added [3H]thymidine, activates LIRn in chloroma cells (figure 7 and table 2) and kills the cells (morphology not shown here but see Rytömaa et ai. 1979). Using a dose estimate of 3 mGy to a chloroma cell nucleus per decay of DNA-bound tritium (see NCRP 1979), we estimate that extensive LIRn activation (followed by death of most or all of the chloroma cells) requires an average DNA dose of a few gray accumulated to the cell nucleus during one cell cycle (i.e. in about 12 h for the subline used here). It should be realized that [3H]TdR concentration added to the Radiation, mobile DNA and programmed death 339 20 10 Unexposed control Spont. activated Ll 4 h after X-rays 24 h Figure 6. Transcriptional activation of LIRn in chloroleukaemia cells by acute X-ray dose (3 C jy). Slot blot analysis of total cytoplasmic RNA (jig given on top of figure) probed with cloned URn(S 5-kb Bam HI fragment from PCB4). culture medium should be related to the cell density: chloroma ceils incorporate approximately 40 per cent of the exogenous tritium from [3H]TdR into DNA (Rytömaa et ai. 1979), and the total activity should not be diluted between too many cells. Compared with the acute X-ray exposure, DN A-bound tritium results in a slow dose rate. This is the most likely reason why transcriptional activity of the LIRn element by 3H does not decrease in chloroma cells at any time after the onset of radiation exposure (results not shown) as it does after acute X-ray exposure. 3.4. Hyperthermia Heat treatment, up to a stress which kills all chloroma cells, does not induce transcriptional activation of the LIRn element; on the contrary, the signal intensity decreases rapidly to about half of the control level (figure 8 and table 2). The results were essentially identical after treatments at 43'C for 3 h and at 44°C for 4 h (cf. figure 8 and table 2, and see §2). Note also that the analysis shown in figure 8 gives the signal intensities for total cellular RNA, i.e. nuclear and cytoplasmic transcripts combined, and therefore the lack of effect cannot be attributed to trapping the LI RNA in the cell nucleus. In accordance with these findings, dying cells do not display the typical nuclear morphology (figure 4C) seen in chloroma cells after spontaneous (Servomaa and Rytömaa 1988) and radiation-induced LIRn activation. 20 10 Unexposed control '4t0. .* **** 7.4 kBq/ml of >H-TdR 37 ,^_ .... Figure 7. Transcriptional activation of lARn in rat chloroleukaemia cells by DNA-bound tritium. ['HjTdR was added to the cultures 24h before isolating total cytoplasmic RNA. Slof blots (/ig RNA given at top of figure) were probed with cloned LIRn (5-5-kb BamHI fragment from p('B4). 340 K. Servomaa and T. Rytömaa unexposed control 4 h after heat shock Spont. activated LI 24 h after heat shock Figure 8. Lack of transcriptional activation of LIRn in chloroleukaemia cells by heat treatment (43 C for 3h). Dot blot analysis of total cellular RNA (nuclear and cytoplasmic combined; ng given on top of figure) probed with cloned LIRn (5-5-kb fragment from pCB4). 4. Discussion We have shown here that, in rapidly growing rat chloroleuka?mia cells, UV light and ionizing radiation induce strong transcriptional activation of the long interspersed repetitive DNA element, LIRn or LINE. Activation of this mobile element is followed by programmed cell death in a sequence of events identical to that seen in the spontaneous LIRn activation occurring at a critical population density (Servomaa and Rytömaa 1988). This type of suicidal cell death, ultimately produced by self-induced lethal mutations through massive retroposition of LI elements (about 300,000 copies) into apparently random locations in the cell genome (Servomaa and Rytömaa 1988), is intriguing, and raises a number of questions. One of the most obvious questions is whether this mechanism of death is generally valid, or is a biological oddity confined to rat chloroleukaemia cells. So far we have no direct experimental evidence to support either alternative. Clearly, however, the idea that a self-destructive mortal programme evolved in an immortal cell line without being basically inherent in normal cells is not easy to accept. We are currently studying this question by inducing apoptosis in a variety of normal cells both in vitro and in vivo. A further question with basic biological relevance concerns the purpose and possible connotations of the activation of the mobile DNA element in chloro leukaemia cells. Apoptosis following radiation may be viewed as an altruistic phenomenon eliminating potentially deleterious cells (Yamada and Ohyama 1988), but in the case of self-destruction of chloroleukaemia cells in vitro this idea is not immediately appealing. On the contrary, the results of the present study suggest a more pessimistic view regarding the activation of a mobile genetic element by two well-established DNA-damaging agents, UV light and ionizing radiation. The observed large-scale activation hints that radiation might, occasionally, also lead to small-scale, non-lethal retroposition of the Li element in the genome, and hence result in potentially hazardous mutations in somatic rells This possibility is not highly speculative because />/ does move in this manner 'spontaneously' also in human genome: the element has been found integrated into the c-myc oncogene in a mammary carcinoma (Morse et al. 1988) and, in some new cases of haemophilia, into the gene encoding factor VIII (Kazazian et al. 1988). Contemporary movement of the LI element in somatic cells has also been observed in experimental animals, Radiation, mobile DNA and programmed death 341 e.g. into the c-myc oncogene in some dog and rat tumours (Katzir et al. 1987, Pear et al. 1988). Although nothing is known of the initiating agents of these retroposi- tions, it is pertinent to recall that several other mobile elements, which are known to play an important role in spontaneous mutations in eukaryotic organisms, show non-!ethal transpositions/retropositions that can be activated by exogenous factors such as radiation (Morawetz 1987). Furthermore, mobile genetic elements which transpose through an RNA intermediate resemble retroviruses (except that they are not known to have an extracellular phase), and endogenous retroviruses have long been known to be activated by radiation in mammalian cells (see Duplan 1977, Schmidt et al. 1985). Taken together, these observations strongly suggest that UV light and ionizing radiation may activate mobile genetic elements even in man, and that genomic transpositions/retropositions of these elements may cause somatic mutations not necessarily followed by readily detectable lethal consequences to the cell (apoptosis). If correct, this speculation adds a new perspective to the mechan ism of radiation-induced biological effects on man (see Rytömaa 1989). One relevant point, directly arising from the present results, concerns molecular mechanisms of the transcriptional activation of the LI element. It appears that the radiation-induced activation of LI is based on, or at least associated with, direct DNA damage. This conclusion is suggested by the finding that non-mutagenic hyperthermia, although lethal to chloroleukaemia cells, does not activate genomic LI and does not kill the cells by phenomic apoptosis. How could one then tie together radiation-induced (DNA-damage-dependent) and spontaneous chloro leukaemia cell deaths, both of which are characterized by ^/-mediated insertional mutations? One possibility is transcriptional activation of the LI element through demethylation (see Cedar 1988), such as might occur during extensive DNA repair and also spontaneously associated with active cell growth. Regarding this, it is tempting to speculate that understanding the initiation of L7-mediated cell death in malignant cells might perhaps have therapeutic consequences. Acknowledgements The authors are grateful to Kirsti Leszczyriski, M.Sc, and Antti Servomaa, Ph.D., for their skilful help in UV and X-irradiation measurements. References BEAVLATON, )., and LocKSMN, R. A., 1982, The relation of programmed cell death to development and reproduction: comparative studies and an attempt at classification. International Review of Cytology, 79, 215-235. CEDAR, H., 1988, DNA rnethylation and gene activity. Cell, 53, 3-4. COM.INS, R. J., VERSCHUER, L. A., HARMON, B. V., PRENTICE, R. 1,., POPE, J. H., and KERR, J. F, R., 1989, Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro. British Journal of Haematology, 71, 343 350. D'AMHROSIO, B., WAITZKIN, S. ].)., WITNEY, F. R.; SAI.EMME, A., and FLHANO, A. V., 1986, Structure of the highly repeated, long interspersed DNA family (LINE or LIRn) of the rat. Molecular and Cellular Biology, 6, 411-424. Di'1'l.AN, J. F. (ed.), 1977, Radiation-induced Leukemogenesis and Related Viruses. Inserm symposium 4 (North-Holland. Amsterdam), FANNING. T., and SINGIH, M., 1987, The UNli-l DNA sequences in four mammalian orders predict proteins that conserve homologies to retrovirus proteins. Nucleic Acids Research, 15, 2251-2260. 342 K. Servomaa and T. Rytömaa GUNN, A., SCRIMGEOUR, D., POTTS, R. C, MACKENZIE, L. A., BROWN, R. A., and SWANSON BECK, J., 1983, The destruction of peripheral-blood lymphocytes by extracorporeal exposure to ultraviolet radiation. Immunology, 50, 477-485. HATTORI, M., KUHARA, S., TAKENAKA, O., and SAKAKI, Y., 1986, LI family of repetitive DNA sequences in primates may be derived from a sequence encoding a reverse transcriptase related protein. Nature, 321,625-628. HENDRY, J. H., POTTEN, C. S., CHADWICK, C, and BIANCHI, M., 1982, Cell death (apoptosis) in the mouse small intestine after low doses: effects of dose-rate, 14-7 MeV neutrons, and 600 MeV (maximum energy) neutrons. International Journal of Radiation Bio logy, 42, b\\-620. KATZIR, N., RECHAVI, G., COHEN, J. B., UNGER, T., SIMONI, F., SEGAL, S., COHEN, D., and GIVOL, D., 1987, 'Retroposon' insertion into the cellular oncogene c-myc in canine transmissible venereal tumor. Proceedings of the National Academy of Sciences, U.S.A., 82, 1054-1058. KAZAZIAN, H. H., WONG, C, YOUSSOUFIAN, H., SCOTT, A. F., PHILLIPS, D. G., and ANTONARAKIS, S. E., 1988, Haemophilia A resulting from de novo insertion of LI sequences represents a novel mechanism for mutation in man. Nature, 332, 164-166. KERR, J. F. R., SEARLE, J., HARMON, B. V., and BISHOP, C. J., 1987, Apoptosis. Perspectives on Mammalian Cell Death, edited by C. S. Potten (Oxford University Press, New York), pp. 93-128. LOEB, D. D., PADGETT, R. W., HARDIES, S. C, SHEHEE, W. R., COMER, M. B., EDGELL, M. H., and HUTCHISON, C. A., 1986, The sequence of a large LIMd element reveals a tandemly repeated 5-end and several features found in retrotransposons. Molecular and Cellular Biology, 6, 168-182. MORAWETZ, C, 1987, Effect of irradiation and mutagenic chemicals on the generation of ADH-2 constitutive mutants in yeast. Significance for the inducibility of Ty transpo sition. Mutation Research, 177, 53-60, 1987. MORSE, B., ROTHERG, P. G., SOUTH, V. J., SPANDORFER, J. M., and ASTRIN, S. M., 1988, Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma. Nature, 333, 87-90. NCRP, 1979, National Council on Radiation Protection and Measurements. Tritium and Other Radionuclide Labeled Organic Compounds Incorporated in Genetic Material, NCRP Report No. 63 (NCRP, Washington). PEAR, W. S., NELSON, S. F., AXELSON, H., WAHLSTRÖM, G.t BAZIN, H., KLEIN, G.r and S'MEGI, J., 1988, Aberrant class switching juxtaposes c-myc with a middle repetitive element (LINE) and an IgH intron in two spontaneously arising rat immunocytomas. Oncogene, 2, 499-507. POTTEN, C. S., 1977, Extreme sensitivity of some intestinal crypt cells to X and y irradiation. Nature, 269, 518-521. ROLFE, M., SPANOS, A., and BANKS, G., 1986, Induction of yeast Ty element transcription by ultraviolet light. Nature, 319, 339 340. RYTÖMAA, T., 1989, Low-dose radiation and cancer. Radiation and Cancer Risk, edited by T. Brustad, F. Langmark and J. B. Reitan (Hemisphere, New York), pp. 3-11. RYTÖMAA, T., and SF.RVOMAA, K., 1986, Identification of a putative growth inhibitor gene in rat chloroleukaemia cells. Biological Regulation of Cell Proliferation, Serono Symposia Publications, vol. 34, edited by R. Baserga, P. Foa, D. Metcalf and E. E. Polli (Raven Press, New York), pp. 93-101, RYTÖMAA, T., SALTEVO, J., and TOIVONEN, H., 1979, Radiotoxicity of tritium-labelled molecules. Biological Implications of Radionuclides Released from Nuclear Industries (IAEA, Vienna), pp. 339-349. SCHMIDT, J,, ERFI.E, V., and MUI.I.F.R, W. A., 1985, Activation of endogenous C-type retroviral genomes by internal alpha-irradiation of mice with 224Radium. Radiation and Environmental Biophysics, 24, 17-25. SCOTT, 1)., and ZAMPETTI-BOSSF.I.FR, F., 1980, The relationship between ceil killing, chromosome aberrations, spindle defects and mitotic delay in mouse lymphoma tells of differential sensitivity to X-ravs. International Journal of Radiation Biology, 37, 33 47. Radiation, mobile DNA and programmed death 343 SERVOMAA, K., and RYTÖMAA, T., 1988, Suicidal death of rat chloroleukaemia cells by activation of the long interspersed repetitive DNA element (LIRn). Cell and Tissue Kinetics, 21, 33-43. SETLOW, R. B., 1974, The wavelengths in sunlight effective in producing skin cancer: a theoretical analysis. Proceedings of the National Academy of Sciences, U.S.A., 71, 3363-3366. STRAND, D. J., and MCDONALD, J. F., 1985, Copia is transcriptionally responsive to environmental stress. Nucleic Acids Research, 13, 4401-4410. UNSCEAR, 1988, Sources, Effects and Risks of Ionizing Radiation. United Nations Scientific Committee on the Effects of Atomic Radiation. 1988 Report of the General Assembly, with Annexes (UN, New York), p. 390. WEEDON, D., SEARLE, J., and KERR, J. F. R., 1979, Apoptosis. Its nature and implications for dermatopathofogy. Ada Dermato-Venereologica, 1, 133-144. WYII.IE, A. H., KERR, j. F. R., and CURRIE, A. R., 1980, Cell death: the significance of apoptosis. International Review of Cytology, 68, 251-306. YAMADA, T., and OHYAMA, H., 1988, Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis). 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