Mutagenesis, Vol. 16, No. 6, 529-537,
November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Chromosome instability induced in the cell progeny of human T lymphocytes irradiated in G0 with 
-rays
1 Department of Biology, University of Padova, Via U. Bassi 58/B, 35020 Legnaro, Padova, Italy 2 INFN-LNL, National Laboratories of Legnaro Via Roma 4, 35020 Legnaro, Padova, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We report the occurrence of chromosome instability in human T lymphocytes irradiated in vitro with
-rays and cultured for several generations before analysis. The delayed effects of -radiation have been evaluated by conventional and molecular (chromosome painting) cytogenetics in preparations obtained from long-term bulk cultures or clonal cultures. The results indicate that the cell progeny of irradiated human T lymphocytes can be characterized by a higher rate of chromosome damage, but this effect depends on the individual donor response to ionizing radiation. Evidence has been collected about a differential involvement of chromosomes 7, 9 and 19 in the induced chromosome rearrangements, and this effect is equally visible as an immediate or delayed response of human T lymphocytes to ionizing radiation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chromosome structural aberrations are among the main genetic alterations induced by ionizing radiation. Depending on dose and Linear Energy Transfer, ionizing radiation can induce even very complex rearrangements among chromosomes and some of them can be transmitted to the progeny as balanced alterations of the karyotype.
Due to the heterogeneous molecular organization of human chromosomes, which are characterized by differential gene density, transcriptional activity and extent of heterochromatic regions, individual chromosomes could show an intrinsically different propensity to breakage events or diverse radiosensitivity. The availability of molecular cytogenetic approaches allowing the labelling of chromosomes with specific fluorescent probes offers the possibility to test this hypothesis. In the last few years, evidence has been obtained about a differential involvement of human chromosomes in breaks and exchanges after radiation exposure (Boei et al., 1997
; Surrallés et al., 1997a; Barquinero et al., 1998; Wojcik and Streffer, 1998; Knehr et al., 1999). However, data are sometimes contrasting and further investigation is necessary.
From the 1980s, it began to become clear from a number of studies that the genetic effects of radiation can be observed in cells that were not directly exposed (for a recent review, see Little, 2000). It has been demonstrated that chromosomal aberrations, point mutations and lethal effects occur at a higher rate in the progeny of irradiated cells with respect to non-irradiated ones, a phenomenon which is known as transmissible genomic instability (Morgan et al., 1996; Little, 2000). In addition, recent data show that genetic damage can be produced by cytoplasmic irradiation (Wu et al., 1999), or can arise in non-irradiated cells neighbouring irradiated ones, an effect known as `bystander effect' (recently reviewed in Grosovsky, 1999; Mothersill and Seymour, 2001). Proposed mechanism(s) underlying the bystander effect include cell to cell communication (Azzam et al., 1998, 2001; Zhou et al., 2000; Belyakov et al., 2001) or the role of extracellular factors (Lehnert and Goodwin, 1997; Narayanan et al., 1997; Iyer and Lehnert, 2000). The relevance of an in vivo bystander effect has been recently addressed (Watson et al., 2000). Furthermore, a relationship between the bystander mechanism and radiation-induced genetic instability has been suggested (Lorimore et al., 1998; Watson et al., 2000). In synthesis, epigenetic mechanisms can play a role in the delayed occurrence of genetic damage after radiation exposure and the initial cell target of radiation does not necessarily coincide with the cells showing the final genetic effects.
The transmission of genome instability has been demonstrated in several experimental systems and by considering different endpoints, such as clonal or non-clonal chromosome aberrations, micronuclei, gene mutations and delayed lethality. Chromosome aberrations of clonal and non-clonal types represent a major manifestation of transmissible instability after radiation exposure (Morgan, 1996; Little, 2000). Chromosome instability has been reported initially after in vitro exposure of murine or human cells to high LET radiation (Kadhim et al., 1992; Sabatier et al., 1992). After these original findings, more data were collected and in particular several aspects such as dependence on dose-, dose-rate and radiation quality were investigated after in vitro or in vivo exposure of several cell types (Holmberg et al., 1993, 1995, 1998; Martins et al., 1993; Kadhim et al., 1994, 1998; Grosovsky et al., 1996; Limoli et al, 1997., 1999; Little et al, 1997.; Ponnaiya et al, 1997.; Watson et al, 1997.; Salomaa et al, 1998.; Belyakov et al, 1999.; Mothersill et al, 2000.). Postnatal transmission of chromosome instability has been also demonstrated in the mouse after fetal irradiation (Devi and Hossain, 2000).
The main concern for a persistent high rate of genetic damage occurring in the progeny of human cells exposed to radiation is represented by the possible increased risk of radiation-induced cancer, which is considered a multistep process, requiring several genetic alterations from initiation to progression. Nevertheless, in spite of the growing evidence about the occurrence of genomic instability as a delayed effect of radiation exposure, the underlying mechanisms are still poorly understood. Indeed, experimental data provided so far do not allow strict comparisons. In particular, the following aspects demand further consideration.
(i)It has been pointed out that the genetic background can be important for the appearance of the delayed response of cells to radiation effects (Kadhim et al, 1994., 1998; Ponnaiya et al, 1997.; Watson et al, 1997.).
(ii)Several studies failed to demonstrate a dose-related response for genomic instability (Kadhim et al, 1992.; Lehnert and Goodwin, 1997; Little et al, 1997.; Lorimore et al, 1998.; Salomaa et al, 1998.; Mothersill et al, 2000.), according to the view that epigenetic mechanisms, instead of directly inducing DNA damage, may be responsible for the appearance of the unstable phenotype. Recently, however, a dose-dependent effect was described, indicating that in a human–hamster hybrid cell line a 3% increase in chromosome instability per Gy of X-rays was obtained after acute exposure (Limoli et al, 1999.).
(iii)Alterations of the classical pathways involved in the restoration of genomic integrity after the initial insult (DNA repair, cell cycle checkpoints, apoptosis) have been proposed to be involved in the acquisition of genomic instability; however, few data have been collected up to now to elucidate this aspect.
Peripheral blood lymphocytes are the reference cells for a large number of assays aimed at evaluating the risk of exposure to environmental agents and for biomonitoring; in particular, these cells have been widely used to describe in vitro and in vivo effects of ionizing radiation. The delayed effects of ionizing radiation in human T lymphocytes have been studied in long-term and clonal cultures set up after in vitro or in vivo exposure to low LET radiation (Holmberg et al, 1993., 1995, 1998; Salomaa et al, 1998.). These studies clearly indicate that chromosome instability may be transmitted along cell generations, and furthermore show how karyotypically abnormal subclones may accumulate further rearrangements during their proliferative growth. In spite of the growing evidence of an heterogeneous involvement of human chromosomes in the radiation-induced rearrangements (Boei et al, 1997.; Surrallés et al, 1997a.; Barquinero et al, 1998.; Wojcik and Streffer, 1998; Knehr et al, 1999.), recurrence of specific chromosomes in the delayed appearance of karyotype abnormalities has been reported only by Martins et al (1993) who found overrepresentation of chromosome 13 in delayed abnormal clones deriving from human skin fibroblasts exposed to heavy ions. In contrast, the alterations described in the clonal progeny of human T lymphocytes (.Holmberg et al, 1993., 1995) and in clones derived from X-irradiated TK6 human B-lymphoblasts (Grosovsky et al, 1996.) did not indicate the existence of chromosome-specific delayed effects of ionizing radiation. However, a preferential susceptibility of pericentromeric heterochromatin has been suggested in the case of complex rearrangements occurring during the proliferation of karyotypically abnormal TK6 clones (Grosovsky et al, 1996.). It should be noted that in all the studies cited above, G-banding was carried out to describe karyotype abnormalities.
In the present study, human T lymphocytes derived from three independent subjects were irradiated in vitro with -rays and cultured for several generations before analysis. Long-term bulk cultures or clonal cell cultures were set up and chromosome analysis was carried out after conventional Giemsa staining or by chromosome painting to evidentiate the possible occurrence of chromosome specific rearrangements. In agreement with previously published data (Holmberg et al, 1993., 1995, 1998; Salomaa et al, 1998.), the present study indicates that chromosome instability can occur in the cell progeny of irradiated human T lymphocytes, but this delayed effect seems to depend on the individual donor response to ionizing radiation. Evidence has been collected about a differential involvement of chromosomes 7, 9 and 19, chosen on the basis of their differential transcriptional activity and chromatin organization (Craig and Bickmore, 1994), in the induced chromosome rearrangements, but this effect is equally visible as an immediate or delayed response to ionizing radiation.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The experiments described in this paper are part of a larger study which includes the analysis of mutational effects at the HPRT locus in the same cell samples used here. The procedure for human T lymphocyte culture and irradiation is described briefly in the following sections; more details can be found in the related paper reporting the HPRT data (Mognato et al, 2001
.).
Cell cultures and irradiation conditions
Peripheral blood lymphocytes were isolated from freshly collected buffy coats of three independent healthy male donors. Buffy coats were anonymously provided by the Blood Hospital Center of Padova; smoking status and age was known only for subject No. 2 (non-smoker, 47 years). Mononuclear cells were isolated by Ficoll-paque, then incubated for 24 h in RPMI 1640 supplemented with 124 U/ml penicillin, 63 µg/ml streptomycin sulfate and 10% heat inactivated fetal calf serum (FCS; Seromed, Germany), at the concentration of 3x106 cells/ml. After removal of adherent monocytes, unstimulated lymphocytes were irradiated at the ` cell' (60Co) of CNR-FRAE (LNL-INFN). Lymphocytes (2x106 cells/ml) were irradiated in 25 or 75 cm2 flasks at doses ranging from 1 to 4 Gy, the dose-rate being 1 Gy/min. Sham irradiated cultures were used for negative controls. Immediately after irradiation, the cells were resuspended in fresh medium and stimulated to proliferation in diverse conditions depending on the specific endpoint (see corresponding sections). In brief, the experiments were designed to quantify for each subject the cell survival, as indicative of the individual immediate response to irradiation. Further evaluation of the immediate response, through chromosome aberration analysis at 50–55 h from irradiation, was considered when a sufficient number of cells was initially isolated. Delayed effects of radiation were studied by chromosome aberration analysis on cell populations derived from long-term bulk cultures harvested at different time intervals from exposure (subject No. 1), or on the clonal progeny of single irradiated cells (subjects No. 2–3).
Determination of cell survival
Lymphocytes, plated 24 h after irradiation in U-bottomed 96-well microtiter plates at 2 cells/well, were grown in RPMI 1640 supplemented with 124 U/ml penicillin, 63 µg/ml streptomycin sulfate, 20% AIM-V (Gibco, UK), 15% defined supplemented bovine serum (DBS; Hyclone, Cramlington, UK), 100 U/ml interleukin-2 (Eurocetus-Chiron, Sienna, Italy), 0.25 µg/ml phytohaemoagglutinin (PHA; Seromed, Berlin, Germany), 2 mM L-Glutamax I (Gibco), 1 mM sodium pyruvate and 50 mM 2-mercaptoethanol. This medium is referred to as Culture Medium (CM; Hou et al, 1999.). TK6 cells lethally irradiated with 40 Gy of X-rays were used as feeder cells (1x104 per well). Two weeks later the cell survival (CS) was determined on the basis of the clonal efficiency in irradiated and control cells (Albertini et al, 1982.).
Long-term bulk cultures
To obtain chromosome preparations for analysis of the immediate effects of irradiation, lymphocytes were resuspended at the concentration of 1x106 cells/ml in RPMI 1640 supplemented with antibiotics, 1% glutamine, 10% FCS and 2.5 µg/ml PHA. Cultures were harvested 50–55 h after stimulation. To study the delayed effects of -radiation, lymphocytes were grown in CM; cultures were started at a concentration of 0.5x106 cells/ml and split every 48 h at the same concentration. Parallel subcultures were harvested at 9, 15, 21 and 28 days after stimulation, the final number of processed cells being 20–30x106.
Clonal cultures
Clonal cell cultures were started as described for cell survival evaluation after 7–9 days of massive culture in CM. This time schedule was necessary because the same clonal cultures were used in the HPRT assay carried out in parellel for the calculation of the cloning efficiency (Mognato et al, 2001.). To expand clonal populations to a minimum of 5x106 cells, clones were collected from microtiter plates, transferred in 24-well plates, then in Petri dishes and finally in culture flasks. Clonal cultures were grown in CM supplemented with 35% T-cell growth factor prepared from a pool of freshly isolated peripheral blood lymphocytes (Hou et al, 1989.). As a starting point, 20–24 clones per experimental condition were initially expanded in multiwell plates. These clones were chosen randomly from microtiter plates from those showing satisfactory proliferation. Clones successfully grown until harvesting were, on average, about one half of the number initially set up.
Chromosome aberration analysis
To accumulate mitotic cells at metaphase, 0.1 µg/ml colcemid (Sigma, Milano, Italy) was added to the cultures during the last 2 h of proliferation and chromosome preparations were obtained by a standard protocol. Slides were stained with 5% Giemsa for conventional cytogenetic analysis. Immediate response to -radiation was studied only for subject No. 2; not less than 100 metaphases from control preparations and for 2 Gy irradiated lymphocytes were scored. For subject No. 1, the same analysis was impaired because of the poor mitotic index at 50 h; for subject No. 3, the initial number of lymphocytes was not sufficient to carry out this task. To evaluate the delayed effects of irradiation, the number of metaphases to be analysed was raised to 300 per experimental point, under the hypothesis that chromosome instability could lead only to small increases of the baseline chromosome aberration frequency. The mitotic index of the harvested cultures allowed us to reach the planned sample size with few exceptions.
Chromosome aberrations were classified as chromosome- or chromatid-types, and for each category fragments and breaks were distinguished from exchanges. Gaps were excluded from the analysis. Percentages of aberrant metaphases in control and treated samples were compared using the G-test. The average number of aberrations per cell was compared between irradiated and non-irradiated samples by applying the Student's t-test.
Fluorescent in situ hybridization with whole chromosome probes (chromosome painting) was applied for a better characterization of induced chromosome damage at the relevant experimental points, as judged from conventional analysis. Probes to chromosomes 7, 9 and 19 were used to verify if preferential involvement of some chromosomes occurred. These chromosomes were selected for analysis according to their differential transcriptional activity and chromatin organization; in particular, chromosome 9 is characterized by a large pericentromeric heterochromatic region; chromosome 7 is of comparable size to chromosome 9, but it lacks large blocks of heterochromatin; chromosome 19 is highly rich in actively transcribed sequences, in spite of its small size (Craig and Bickmore, 1994). Probes obtained from microdissected whole chromosomes were purchased from Li Star Fish, Carugato, Milano, Italy. Probes to chromosomes 7 and 19 were labelled with biotin and detected by Cy3-avidin (Sigma). Chromosome 9 probe was digoxigenin-labelled and detected by anti-dig antibodies conjugated to fluorescein (Roche Biochemicals, Germany). The procedure for chromosome painting was carried out according to the manufacturer's instructions.
Chromosome painting analysis was carried out under a Zeiss Axioskop microscope equipped with filters for UV, blue and green light, and with a triple band pass filter for the simultaneous vision of DAPI staining, Cy3 and fluorescein. The number of analysed metaphases was variable, according to the sample availability and the total number of aberrations expected on the basis of conventional analysis. The PAINT nomenclature (Tucker et al, 1995.) was used to classify the observed aberrations. Chromosome aberrations involving unlabelled chromosomes were recorded throughout. The frequencies of rearrangements involving painted chromosomes (colour junctions) were weighted upon the whole genome (genomic junctions) by applying the formula provided by Lucas et al (1992). The relative size of chromosomes under evaluation is that reported by Morton (1991)..
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The individual response to
irradiation of lymphocytes deriving from the three healthy donors used in this study was assessed as cell survival in a clonogenic assay carried out in a parallel study (Mognato et al, 2001.). The data concerning the three individuals included in the present cytogenetic analysis are summarized in Table I: subject No. 1 appears to be the most sensitive to irradiation, the cell survival corresponding to 19.3% of the control at 2 Gy. At the same dose, we observed 43% survival for subject No. 2, while for subject No. 3 the survival corresponded to 65% of the control. The complete survival curve calculated with individual data coming from these and other subjects is reported elsewhere (Mognato et al, 2001.). The average value estimated at 2 Gy is 44.7%, the standard deviation corresponding to 16.5%.
Table II shows the results obtained after chromosome aberration analysis in lymphocyte cell populations derived from subject No. 1 and grown in bulk cultures after irradiation. Parallel cultures were set up and harvested at different time intervals (9, 15, 21 and 28 days) after irradiation with 1, 2 and 3 Gy. Matched control cultures were considered. Chromosome preparations obtained 9 days after 3 Gy irradiation were not amenable for cytogenetic analysis because of the poor mitotic index. The same was found 28 days after irradiation with different doses and, therefore, this late time interval was not further considered for cytogenetic analysis. In the non-irradiated population the frequency of aberrant cells ranged from 2.93 to 5.63%. After 1 Gy -irradiation a statistically significant increase of aberrant metaphases with respect to the control was detected only at the 9 day time interval (P < 0.005; 2x2 G-test). A significant increase of aberrant cells was observed up to 15 days from exposure to 2 Gy -rays (P < 0.05), while significant differences were found in cells after exposures of 3 Gy up to 21 days (P < 0.05). A similar pattern was observed by taking into account the mean number of aberrations per cell. Statistical comparisons were carried out by Student's t-test and confirmed the results from the G-test with the only exception of the sample analysed 21 days after a 3 Gy dose. The chromosome number distribution did not show variation among the samples analysed. Figure 1 exemplifies this situation by comparing chromosome number distributions for control cultures and cultures grown after a dose of 2 Gy -irradiation.
|
|
Table III
summarizes the results of chromosome aberration analysis carried out on lymphocytes derived from subject No. 2 and cultured for 52 h after irradiation (2 Gy). An 8-fold increase in the baseline frequency of aberrant metaphases was observed (P < 0.001); the mean number of aberrations per cell rose from 0.04 to 0.55 (P < 0.001).
|
The delayed effects of
-radiation in lymphocytes from the same subject were assayed by chromosome analysis in clonal cultures and the results are shown in Table IV. These clones were harvested 50–55 days after irradiation. Only nine of the clones could be analysed (two obtained from non-irradiated lymphocytes, seven after 2 Gy irradiation), the remainder showed a low mitotic index and were discarded. A 2x2 G-test was carried out to compare the proportion of aberrant metaphases observed in each clone derived from irradiated cells with the pooled data obtained from clones No. 1 and 2 derived from non-irradiated cells. A significant increase in the frequency of aberrant metaphases was found for clone Nos 4 and 8 (P < 0.001). The pooled data coming from clones derived from irradiated cells were also found to differ significantly (P < 0.001) from the pooled control data. The large majority of chromosome aberrations observed in the clonal cultures derived from subject No. 2 appeared to be non-transmissible (Table IV). Statistical comparisons between the average number of aberrations per cell in the control sample and in each clone derived from irradiated cells was carried out by Student's t-test. The results demonstrated that four clones derived from irradiated cells (Nos 4, 5, 8 and 9) carry a higher number of aberrations per cell than control clonal cultures (Table IV). The chromosome number distribution relative to each clone always showed the normal diploid modal value and the overlapping to control distributions was clear (data not shown).
|
Table V
shows data from chromosome aberration analysis in clonal cultures obtained from subject No. 3, whose lymphocytes were irradiated with doses of 0.5, 1 and 2 Gy. Clonal cultures set up from irradiated or non-irradiated cells were harvested 35 days after exposure, but only 10 of them provided chromosome spreads that could be analysed and in general the proliferative activity of these populations was low. No differences were observed between clones derived from irradiated cells with respect to the pooled data coming from clones Nos 1–3, representing control samples. No deviations from the normal chromosome number distribution were observed for this group of clones (data not shown).
|
To evaluate if the preferential occurrence of chromosome breaks in some regions of the chromosome is involved in the onset of chromosome instability, we carried out chromosome painting analysis in a number of samples. Lymphocytes derived from subject No. 1 and grown 9 days after
irradiation (2 Gy) were analysed by chromosome painting with probes to chromosomes 7 and 9. 563 chromosome spreads were scored and the results concerning the involvement of the two labelled chromosomes are given in Table VI. Chromosome 9 appeared to be involved 1.7-fold more frequently than chromosome 7 in the observed rearrangements. The large majority of exchanges were translocations, while dicentrics were seldom observed. All the aberrations involving unlabelled chromosomes were recorded in the course of the molecular cytogenetic analysis. We observed 28 breaks of the chromatid type, 13 of chromosome type and 10 dicentrics. The dicentric frequency, calculated as the total number of labelled plus unlabelled dicentrics, appeared only slightly higher than evaluated by Giemsa analysis.
|
From the lymphocytes of subject No. 2, we analysed by chromosome painting the preparations harvested after 52 h of culture (0 and 2 Gy
irradiation) and the preparations from clones Nos 3, 4 and 8 (all derived from irradiated cells). The results obtained after analysis of 52 h preparations are shown in Table VII for the irradiated sample only, as in the control we observed only one metaphase with a break involving chromosome 7 and one with a break involving chromosome 9, on a total of 440 scored cells. Again we observed a preferential representation of chromosome 9 with respect to chromosome 7, equal to a factor 1.4. Translocations were about twice the number of dicentrics for both chromosomes under study. Apart from the chromosome spreads carrying rearranged labelled chromosomes, at this short time after irradiation, we observed on the total of 600 scored metaphases a number of aberrations involving other chromosomes; in particular, 65 dicentrics but only 2 rings. The percentage of dicentrics observed after fluorescent microscopy appeared in very good agreement with that obtained after Giemsa staining (14 versus 11.8%).
|
From the same preparations, a sample of chromosome spreads was analysed with a probe for chromosome 19. This small chromosome represents only 2.15% of the genome, but on a total of 150 scored spreads we found 14 colour junctions, which in percentage terms corresponds to 9.3%. After weighting this value to the whole genome, we obtain 221.03 genomic junctions. In conclusion chromosome 19 appears highly represented in chromosome rearrangements, in spite of its reduced size.
Concerning the clonal samples, the analysis of 364 metaphases from clone 8 revealed only two aberrations involving labelled chromosomes: a t(Ba) for chromosome 9 and an ace(b) for chromosome 7. Clone No. 3 gave 915 analysable metaphases and 13 of them showed aberrations involving labelled chromosomes. From clone No. 4, 740 metaphases were analysed and 17 aberrations were found to contain labelled chromosomes. The results from the analysis of clones Nos 3 and 4 are summarized in Tables VIII and IX. All the observed aberration were non-clonal, as they appeared as single events in the clonal progeny of single cells. The total number of rearrangements observed was judged too low to make inferences about a differential representation of chromosomes 7 and 9 in chromosome aberrations.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study we described the long-term effects of
-rays on peripheral blood T-lymphocytes irradiated in vitro before proliferation. Although several studies over the last decade documented the potential of ionizing radiation in inducing genomic instability, and in particular chromosome instability, further clarification of many aspects of the phenomenon is necessary to understand its molecular basis
Several critical aspects have been highlighted by previous studies of chromosome instability. First, a minimum number of cell generations may be necessary to reveal this effect (Ponnaiya et al, 1997.; Kadhim et al, 1998.). Secondly, the role of the administered dose in the induction of chromosome instability is unclear (Morgan et al, 1996.; Limoli et al, 1999.). Finally, the genotype of irradiated cells may play a role in the manifestation of chromosome instability (Ponnaiya et al, 1997.; Watson et al, 1997.; Kadhim et al, 1998.). Having these points in mind, in the first part of the study we tried to monitor the temporal variation pattern of the aberration frequency in bulk cultures of lymphocytes after several doses of irradiation. We found that the frequency of aberrations in the bulk cultures decreased along with time, reaching the control value in a dose-related manner: in cultures irradiated with 1 Gy, the chromosome aberration frequency significantly differed from the control only at 9 days after irradiation, it was still higher than control at 15 days in cultures irradiated with 2 Gy, and finally a difference was detectable at the longest time interval tested (21 days) after 3 Gy irradiation. This dose-related effect however does not correspond to a simple dose–effect relationship as observed by Limoli et al. (1999); for example, comparable increases of about 2-fold the control frequency were found at the 9 day time interval for samples irradiated with 2 or 3 Gy. It is possible that the balance among different immediate effects of irradiation, including DNA damage and cell lethality, account for the temporal pattern observed at different doses. Although in the case of lymphocytes from subject No. 1, the chromosome aberration frequency was studied in bulk cultures and not in the progeny of individual cells, the effect detected at delayed time intervals can be easily interpreted as an indirect effect of irradiation. First a very large proportion of the observed rearrangements represent non-transmissible unstable aberrations, such as chromatid/chromosome fragments, or chromatid interchanges. Therefore we must assume that the observed alterations occurred just before the harvesting of the cell culture. Secondly, we applied chromosome painting and again we observed a number of aberrations, some of them representing chromosome translocations, which are not evident with conventional cytogenetic analysis. Translocations are viable rearrangements and could accumulate in the cell progeny. However, we did not find any evidence supporting the presence of clonal karyotype alterations, as far as the chromosomes under investigation were concerned. All the observed aberrations appeared as unique events in the population and over time.
Next we focused on the analysis of clonal cell progeny after a single irradiation dose (2 Gy). A number of clones carried a higher chromosome aberration frequency with respect to clones derived from non-irradiated cells. As in the case of long-term bulk cultures derived from the previous subject, all the chromosome alterations observed in clonal cultures from subject No. 2 belonged to the unstable type, indicating their delayed onset. Chromosome painting analysis did not indicate any contribution of transmissible aberrations in the origin of analysed clones (i.e. a marker rearrangement). For the above reasons, the observed chromosome aberrations must be considered as occurring de novo during clonal proliferation. Remarkably, the chromosome number appeared quite stable in all clonal populations studied from subject No. 2, as well as in the massive populations cultured from lymphocytes of subject No. 1.
From a third subject, we considered the potential of different doses with respect to the transmission of chromosome instability in the progeny of irradiated T lymphocytes. Unfortunately, in this case we were not able to detect unstable clones among the very few clones we successfully expanded from this subject. There are several different possible explanations for the lack of transmission of instability in this last subject: first, if the ability to observe chromosome instability is a dose-dependent effect, as it would seem from the results collected on subject No. 1, we could have probably disregarded the event because of the limited sample size. Alternatively, the absence of unstable clones could be due to the shortest time interval considered for clone harvesting with respect to that applied in the case of subject No. 2. Indeed, it has been proposed that a minimum number of cell generations may be necessary for the unstable phenotype to appear (Ponnaiya et al, 1997.; Kadhim et al, 1998.). The present data obtained from long-term bulk cultures of lymphocytes (subject No. 1) indicate, on the other hand, that chromosome instability can be observed even at 9 days after irradiation, a time interval comprising a very limited number of cell doublings. Although the significance and the biological meaning of a temporal pattern for the emergence of an unstable phenotype is puzzling, it is hard to address this question by working with human T lymphocytes, because of the limited reproducibility of the cell culture conditions: lymphocytes of different individuals can be differentially stimulated to proliferation by the growth medium; furthermore, the medium itself can only be produced in finite batches. Consequently, the long-term cultures of human T lymphocytes tend to cease their proliferation at variable time intervals. In the case of subject No. 3, it was in fact not possible to maintain clones as long as for subject No. 2, a complicating factor for the interpretation of the results. The last important aspect to be considered as an explanation for the different behaviour of lymphocytes from subjects No. 2 and No. 3 is shown by their differential response to irradiation, as indicated by the cell survival data at 24 h from irradiation. Subject No. 3 showed a particularly high cell survival value (Table I), with respect to subjects No. 1 and 2 and with respect to the value obtained for this dose by fitting the whole survival curve (based on the present three and two additional subjects; Mognato et al, 2001.). It is reasonable to postulate that this different response can account for the lack of induction of delayed effects of irradiation, confirming the importance of the genetic background as already suggested (Ponnaiya et al, 1997.; Watson et al, 1997.; Kadhim et al, 1998.).
|
Previous evidence concerning the ability of low LET ionizing radiation to induce chromosome instability in peripheral blood lymphocytes has been provided by analysing karyotype alterations in the long-term and clonal cultures of T lymphocytes (Holmberg et al, 1993.
, 1995, 1998; Salomaa et al, 1998.). The present data confirm that low LET radiation induces chromosome instability in primary human lymphocytes and suggest that the possibility to observe this delayed effect can depend on the administered dose and interindividual variability. Interestingly, the possibility that chromosome instability effects after irradiation of resting lymphocytes can be influenced by the experimental conditions for mitogen stimulation has been ruled out (Holmberg et al, 1998.).
Since many studies indicate that the preferential involvement of some chromosomes can be observed after exposure to ionizing radiation (Boei et al, 1997.; Surrallés et al, 1997a.; Barquinero et al, 1998.; Wojcik and Streffer, 1998; Knehr et al, 1999.), the behaviour of chromosomes 7, 9 and 19 was studied here. Chromosomes 7 and 9 are comparable in size and gene density; however, chromosome 9 is characterized by a large heterochromatic region. Heterochromatin has been reported to be especially prone to chromosome breakage (Eastmond et al, 1994.; Rupa et al, 1995.; Surrallés et al, 1997b.; Puerto et al, 2000.). In fact, we observed in this study that chromosome 9 can be over-represented in the observed rearrangements by a factor of 1.5–2.0 with respect to chromosome 7. This observation was obtained either after cytogenetic analysis at 52 h from irradiation (immediate effects of radiation) or in preparations harvested 9 days after irradiation (delayed effects of irradiation). Thus, it does not seem that chromosome 9 is particularly sensitive to radiation damage itself, while it appears highly prone to chromosome breakage. The observation that there is a higher representation of chromosome 19 among the observed aberrations induced by -rays is still preliminary; however, it suggests that besides silent regions such as the heterochromatin, even highly expressed sequences as those belonging to chromosome 19 can frequently participate in chromosome rearrangements.
Although many questions still remain open and probably require different experimental strategies to obtain an answer, the results collected up to now confirm the relevance of studying the modalities of chromosome instability induction in clonal cultures of primary human cells. To improve our perspective, we are at present collecting data about the response of human T lymphocytes to low energy protons. We aim by taking this view to understand the role of radiation quality (in particular as far as LET differences are concerned) on the onset of chromosome instability in human lymphocytes.
|
Acknowledgments |
|---|
We are grateful to Dr. S. Lora (LNL-INFN) for his collaboration in irradiation experiments. This study has been granted by the University of Padova (research grant 1998).
|
Notes |
|---|
4 To whom correspondence should be addressed. Email: antonella.russo{at}uninsubria.it
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Albertini,R.J., Castle,K.L. and Borcherding,W.R. (1982) T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood. Proc. Natl Acad. Sci. USA, 79, 6617–6621.
Azzam,E.I., de Toledo,S.M., Gooding,T. and Little,J.B. (1998) Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of 
particles. Radiat. Res., 150, 497–504.[Web of Science][Medline]
Azzam,E.I., de Toledo,S.M. and Little,J.B. (2001) Direct evidence for the participation of gap junction-mediated intercellular communication in the transmision of damage signals from -particle irradiated to nonirradiated cells. Proc. Natl Acad. Sci. USA, 98, 473–478.
Barquinero,J.F., Knehr,S., Braselmann,H., Figel,M. and Bauchinger,M. (1998) DNA-proportional distribution of radiation-induced chromosome aberrations analysed by fluorescence in situ hybridization painting of all chromosomes of a human female karyotype. Int. J. Radiat. Biol., 74, 315–323.[Web of Science][Medline]
Belyakov,O.V., Prise,K.M., Trott,K.R. and Michael,B.D. (1999) Delayed lethality, apoptosis and micronucleus formation in human fibroblasts irradiated with X-rays or -particles. Int. J. Radiat. Biol., 75, 985–993.[Web of Science][Medline]
Belyakov,O.V., Malcolmson,A.M., Folkard,M., Prise,K.M. and Michael,B.D. (2001) Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts. Br. J. Cancer, 84, 674–679.[Web of Science][Medline]
Boei,J.J.W.A., Vermeulen,S. and Natarajan,A.T. (1997) Differential involvement of chromosomes 1 and 4 in the formation of chromosomal aberrations in human lymphocytes after X-irradiation. Int. J. Radiat. Biol., 72, 139–145.[Web of Science][Medline]
Craig,J.M. and Bickmore,W.A. (1994) The distribution of CpG islands in mammalian chromosomes. Nature Genet., 7, 376–381.[Web of Science][Medline]
Devi,P.U. and Hossain,M. (2000) Induction of chromosomal instability in mouse hemopoietic cells by fetal irradiation. Mutat. Res., 456, 33–37.[Web of Science][Medline]
Eastmond,D.A., Rupa,D.S. and Hasegawa,L. (1994) Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes. Mutat. Res., 322, 9–20.[Web of Science][Medline]
Grosovsky,A.J., Parks,K., Giver,C.R. and Nelson,S.L. (1996) Clonal analysis of delayed karyotypic abnormalities and gene mutations in radiation-induced genetic instability. Mol. Cell. Biol., 16, 6252–6262.[Abstract]
Grosovsky,A.J. (1999) Radiation-induced mutations in unirradiated DNA. Proc. Natl Acad. Sci. USA, 96, 5346–5347.
Holmberg,K., Fält,S., Johansson,A. and Lambert,B. (1993) Clonal chromosome aberrations and genome instability in X-irradiated human T-lymphocyte cultures. Mutat. Res., 286, 321–330.[Web of Science][Medline]
Holmberg,K., Meijer,A.E., Auer,G. and Lambert,B. (1995) Delayed chromosomal instability in human T-lymphocyte clones exposed to ionizing radiation. Int. J. Radiat. Biol., 68, 245–255.[Web of Science][Medline]
Holmberg,K., Meijer,A.E., Harms-Ringdahl,M. and Lambert,B. (1998) Int. J. Radiat. Biol., 73, 21–34.
Hou,S.M., Holmberg,K., Lambert,B. and Einhorn,N. (1989) Hprt mutations and karyotype abnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients. Mutat. Res., 210, 353–358.[Web of Science][Medline]
Hou,S.M., VanDam,F.J., deZwart,F. et al. (1999) Validation of the human T-lymphocyte cloning assay-ring test report from the EU concerted action on HPRT mutation (EUCAHM). Mutat. Res., 431, 211–221.[Web of Science][Medline]
Kadhim,M.A., Macdonald,D.A., Goodhead,D.T., Lorimore,S.A., Marsden,S.J. and Wright,E.G. (1992) Transmission of chromosomal instability after plutonium -particle irradiation. Nature, 355, 738–740.[Medline]
Kadhim,M.A., Lorimore,S.A., Hepburn,M.D., Goodhead,D.T., Buckle,V.J. and Wright,E.G. (1994) Alpha-particle-induced chromosomal instability in human bone marrow cells. Lancet, 344, 987–988.[Web of Science][Medline]
Kadhim,M.A., Marsden,S.J. and Wright,E.G. (1998) Radiation-induced chromosomal instability in human fibroblasts: temporal effects and the influence of radiation quality. Int. J. Radiat. Biol., 73, 143–148.[Web of Science][Medline]
Knehr,S., Huber,R., Braselmann,H., Schraube,H. and Bauchinger,M. (1999) Multicolour FISH painting for the analysis of chromosomal aberrations induced by 220 kV X-rays and fission neutrons. Int. J. Radiat. Biol., 75, 407–418.[Web of Science][Medline]
Iyer,R. and Lehnert,B.E. (2000) Factors underlying the cell growth-related bystander responses to particles. Cancer Res., 60, 1290–1298.
Lehnert,B.E. and Goodwin,E.H. (1997) Extracellular factor(s) following exposure to particles can cause sister chromatid exchanges in normal human cells. Cancer Res., 57, 2164–2171.
Limoli,C.L., Kaplan,M.I., Corcoran,J., Meyers,M., Boothman,D.A. and Morgan,W.F. (1999) Chromosomal instability and its relationship to other end points of genomic instability. Cancer Res., 57, 5557–5563.
Limoli,C.L., Corcoran,J.J., Milligan,J.R., Ward,J.F. and Morgan,W.F. (1999) Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation. Radiat. Res., 151, 677–685.[Web of Science][Medline]
Little,J.B., Nagasawa,H., Pfenning,T. and Vetrovs,H. (1997) Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of X-rays and particles. Radiat. Res., 148, 299–307.[Web of Science][Medline]
Little,J.B. (2000) Radiation carcinogenesis. Carcinogenesis, 21, 397–404.
Lorimore,S., Kadhim,M., Pocock,D., Stevens,D., Papworth,D., Goodhead,D. and Wright,E. (1998) Chromosomal instability in the descendants of unirradiated surviving cells after -particle irradiation. Proc. Natl Acad. Sci. USA, 95, 5730–5733.
Lucas,J.N., Awa,A., Straume,T., Poggensee,M., Kodama,Y., Nakano,M., Ohtaki,K., Weier,H.U., Pinkel,D., Gray,J. and Littlefield,G. (1992) Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation. Int. J. Radiat. Biol., 62, 53–63.[Web of Science][Medline]
Holmberg,K., Meijer,A.E., Auer,G. and Lambert,B. (1995) Delayed chromosomal instability in human T-lymphocyte clones exposed to ionizing radiation. Int. J. Radiat. Biol., 68, 245–255.
Martins,M.B. Sabatier,L., Ricoul,M., Pinton,A. and Dutrillaux,B. (1993) Specific chromosome instability induced by heavy ions: a step towards transformation of human fibroblasts? Mutat. Res., 285, 229–237.[Web of Science][Medline]
Mognato,M., Ferraro,P., Canova,S., Sordi,G., Russo,A., Cherubini,R. and Celotti,L. (2001) Analysis of mutational effects at the HPRT locus in human G0 phase lymphocytes irradiated in vitro with rays. Mutat. Res., 474, 147–158.[Web of Science][Medline]
Morgan,W.F., Day,J.P., Kaplan,M.I., McGhee,E.M. and Limoli,C. (1996) Genomic instability induced by ionizing radiation. Radiat. Res., 146, 247–258[Web of Science][Medline]
Morton,N.E. (1991) Parameters of the human genome. Proc. Natl Acad. Sci. USA, 88, 7474–7476.
Mothersill,C., Kadhim,K.A., O'Reilly,S., Papworth,D., Marsden,S.J., Seymour,C.B. and Wright,E.G. (2000) Dose- and time-response relationships for lethal mutations and chromosomal instability induced by ionizing radiation in an immortalized human keratinocyte cell line. Int. J. Radiat. Biol., 76, 799–806.[Web of Science][Medline]
Mothersill,C. and Seymour,C. (2001) Radiation-induced bystander effects: past history and future directions. Radiat. Res., 155, 759–767.[Web of Science][Medline]
Nagasawa,H. and Little,J.B. (1999) Unexpected sensitivity to the induction of mutations by very low doses of -particle radiation: evidence for a bystander effect. Radiat. Res., 152, 552–557.[Web of Science][Medline]
Narayanan,P.K., Goodwin,E.H. and Lehnert,B.E. (1997) -Particles initiate biological production of superoxide anions and hydrogen peroxide in human cells. Cancer Res., 57, 2164–2171.
Ponnaiya,B., Cornforth,M.N. and Ullrich,R.L. (1997) Radiation-induced chromosomal instability in BALB/c and C57BL/6 mice: the difference is as clear as black and white. Radiat. Res., 147, 121–125.[Web of Science][Medline]
Puerto,S., Marcos,R., Ramírez,M.J., Creus,A., Boei,J.J.W.A., Meijers,M., Natarajan,A.T. and Surrallés,J. (2000) Induction, processing and persistence of radiation-induced chromosomal aberrations involving hamster euchromatin and heterochromatin. Mutat. Res., 469, 169–179.[Web of Science][Medline]
Rupa,D.S., Hasegawa,L. and Eastmond,D.A. (1995) Detection of chromosomal breakage in the 1cen-1q12 region of interphase human lymphocytes using multicolour fluorescence in situ hybridization with tandem DNA probes. Cancer Res., 55, 640–645.
Sabatier,L., Martins,B. and Dutrillaux,B. (1992) Chromosomal instability. Nature, 357, 548.[Medline]
Salomaa,S., Holmberg,K., Lindholm,C., Mustonen,R., Tekkel,M., Veidebaum, T. and Lambert,B. (1998) Chromosomal instability in in vivo radiation exposed subjects. Int. J. Radiat. Biol., 74, 771–779.[Web of Science][Medline]
Surrallés,J., Sebastian,S. and Natarajan,A.T. (1997a) Chromosomes with high gene density are preferentially repaired in human cells. Mutagenesis, 12, 437–442.
Surrallés,J., Darroudi,F. and Natarajan,A.T. (1997b) Low level of DNA repair in human chromosome I heterochromatin. Genes Chromsom. Cancer, 20, 173–184.[Web of Science][Medline]
Tucker,J.D., Morgan,W.F., Awa,A.A., Bauchinger,M., Blakey,D., Cornforth,M.N., Littlefield,L.G., Natarajan,A.T. and Shasserre,C. (1995) A proposed system for scoring structural aberrations detected by chromosome paintin. Cytogenet. Cell Genet., 68, 211–221.[Web of Science][Medline]
Watson,G.E., Lorimore,S.A., Clutton,S.M. and Wright,E.G. (1997) Genetic factors influencing -particle-induced chromosomal instability. Int. J. Radiat. Biol., 71, 497–503.[Web of Science][Medline]
Watson,G.E., Lorimore,S.A., Macdonald,D.A. and Wright,E.G. (2000) Chromosomal instability in unirradiated cells induced in vivo by a bystander effect of ionizing radiation. Cancer Res., 60, 5608–5611.
Wojcik,A. and Streffer,C. (1998) Comparison of radiation-induced aberration frequencies in chromosomes 1 and 2 of two human donors. Int. J. Radiat. Biol., 74, 573–581.[Web of Science][Medline]
Wu,L.J., Randers-Pehrson,G., Xu,A., Waldren,C.A., Geard,C.R., Yu,Z.L. and Hei,T.K. (1999) Targeted cytoplasmic irradiation with particles induces mutations in mammalian cells. Proc. Natl Acad. Sci. USA, 96, 4959–4964.
Zhou,H., Randers-Pehrson,G., Waldren,C.A., Vannais,D., Hall,E.J. and Hei,T.K. (2000) Induction of a bystander mutagenic effect of particles in mammalian cells. Proc. Natl Acad. Sci. USA, 97, 2099–2104.
Received on March 13, 2001; accepted on July 23, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||