Correlation between cell reproductive death and chromosome ...

int. j. radiat. biol 1999, vol. 75, no. 3, 293 ± 299

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells ´ W†‡, C. VAN BREE†§, J. B. A. KIPP†, N. A. P. FRANKEN†*, P. RUURS†, G. LUDWIKO F. DARROUDI¶ and G. W. BARENDSEN† (Received 21 August 1998; accepted 22 October 1998) Abstract. Purpose: To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with c-rays. Method: Linear–quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with  uorescence in situ hybridization in chromosomes X and 2. Results: DiÚ erences in the LQ parameters a and b of both nonsensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with nonsensitized cells was observed. The a-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the b coeÝ cient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no signiŽ cant diÚ erence in a was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher b was found for the sensitized X-chromosome compared to this value for chromosome 2. Conclusions: It can be concluded that the linear–quadratic analysis of dose–response relationships oÚ ers insights into the correlation between cell survival and induction of exchanges in nonsensitized and radiosensitized cells.

1. Introduction Radiation damage to mammalian cells expressed as reproductive death and chromosome aberrations is determined by many factors, including intrinsic genetic characteristics, repair proŽ ciency, chromatin conformation, cell-cycle stage and metabolic or *Author for correspondence: e-mail: [email protected] †Department of Radiotherapy, ‡Centre for Microscopical Research, Department of Cell Biology and Histology, and §Department of Internal Medicine, Academic Medical Centre, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands. ¶MGC, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden Medical Centre and J. A. Cohen Institute of Radiopathology and Radiation Protection, Sylvius Laboratories, Wasseraavsewg 72, 2333 AL Leiden, The Netherlands.

hypoxic conditions. Incorporation of halogenated prymidines (HP) into cellular DNA reduces clonogenic cell survival after ionizing radiation (Iliakis et al. 1989, 1991, Franken et al. 1997a,b, van Bree et al. 1997). This enhancement of cell reproductive death can be compared with the observed increase in the number of chromosome aberrations to evaluate whether similar or diÚ erent mechanisms are involved (Lawrence et al. 1990, Ling and Ward 1990, Iliakis et al. 1992). Increases of DNA double-strand breaks (dsb) after incorporation of HP have been measured with pulsed-Ž eld gel electrophoresis (Latz et al. 1994, Lawrence et al. 1995), and with the neutral or alkaline elution techniques (Lawrence et al. 1990, Ling and Ward 1990, Limoli and Ward 1993, Jones et al. 1995). These results have provided valuable information on the biochemical mechanisms of these responses and on repair kinetics, and for quantitative comparisons of the enhancement of biological eÚ ects by HP. The degree of sensitization by HP shows diÚ erences with respect to the endpoints investigated. Moreover, variability has been reported for the same endpoint studied for diÚ erent chromosomes. Concerning the radiosensitivity of individual human chromosomes, diÚ erent data were reported. Wilt et al. (1994) studied the in uence of bromodeoxyuridine (BrdUrd) on the induction of chromosomal aberrations in two diÚ erent chromosomes, #1 and #4, of a human carcinoma cell line. A lower susceptibility of chromosome 4 compared with chromosome 1 was evident. In contrast, in human lymphocytes, Boei et al. (1997) observed an over-involvement of chromosome 4 with respect to the frequency of reciprocal exchanges compared with chromosome 1. Generally, all the above-mentioned experiments were based on one donor and one dose and therefore further investigation is required. Fluorescence in situ hybridization (FISH) allows the assessment of dose–eÚ ect relationships for cytogenetic damage to individual chromosomes. Exchange frequencies as a function of the dose have

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been reported for human lymphocytes and several human tumour cell lines (Lucas et al. 1992, Natarajan et al. 1992, Bauchinger et al. 1993, Ferna´ ndez et al. 1995, Coco-Martin et al. 1996, Simpson and Savage 1996, Kovacs et al. 1997, Virsik-Peuckert et al. 1997, Franken et al. in press 1999, Lindholm et al. 1998). For a comparison of dose–eÚ ect relationships obtained with X-rays, the linear–quadratic (LQ) description is the most suitable because it allows analysis of the eÚ ectiveness of small doses and large doses separately. Based on biophysical considerations, it is suggested that the linear term represents damage induced by a single ionizing particle and its secondaries, while the quadratic term represents damage due to interaction of lesions induced by diÚ erent ionizing particles. The parameters can be discussed in terms of speciŽ c mechanisms of cell inactivation by radiation (Barendsen 1990, 1997). The frequency of lesions as a function of the dose D can thus be described by F(D) = aD+bD2 (Barendsen 1982, 1990, 1997). For this analysis, data on dose– response relationships for an adequate range of doses are required. From data for a single dose, enhancement factors deŽ ned as ratios of doses required to obtain a speciŽ ed eÚ ect cannot be derived reliably, except for cases of linear increases of eÚ ects as a function of the dose. Virsik-Peuckert et al. (1997) observed diÚ erences in the parameters a and b in the LQ analysis for three diÚ erent chromosomes (#4, #7 and #9) of lymphocytes obtained from a healthy donor. In order to contribute to the information on diÚ erences in eÚ ectiveness of radiosensitization by HP for diÚ erent endpoints and between diÚ erent chromosomes, the authors have investigated the induction of cell reproductive cell death and exchanges (using whole-chromosome-speciŽ c DNA probes for chromosome #2 and chromosome X and FISH) by c-rays in the human tumour cell line designated as SW-1573. The ultimate aim of an increased insight is to evaluate whether tumours can be sensitized more than normal tissues and whether chromosome damage can be used to predict susceptibility to cell death. In the present communication, parameters of the LQ model were analysed from dose–eÚ ect relationships for the induction of cell death and for exchanges in the X-chromosome and in chromosome #2 of SW-1573 cells, and the changes induced by radiosensitization with IdUrd were evaluated. The aim of the comparison with cell reproductive death was to assess whether the parameters a and b derived for these endpoints could be diÚ erently aÚ ected and thus information obtained on diÚ erences in the

mechanisms involved in the expression of these types of damage. 2. Materials and methods 2.1. Cell culture The human squamous lung carcinoma cell line SW-1573 was grown at 37ß C as monolayers in 75 cm2 tissue culture  asks (Costar Europe LTD, Badhaevedorp, The Netherlands) in Leibowitz-15 medium (L-15; GIBCO-BRL Life Technologies, Breda, The Netherlands) supplemented with 10% foetal bovine serum and 2 mm glutamine. The L-15 medium does not require CO2 atmosphere in the incubator. The doubling time of the cells during exponential growth is 22–24 h (Haveman et al. 1995). For experiments, the cells were incubated for 72 h in medium containing 0 or 4 mm of iodo-deoxyuridine (IdUrd) (Sigma, St Louis, MO, USA). During this incubation period the medium was not changed. Cells were irradiated in plateau phase and either used for clonogenic assay or metaphase preparation. The cell-cycle distribution was monitored by  ow cytometry and at the time of irradiation over 95% of the cells were in G0+G1 phase. 2.2. Determination of percentage thymidine replacement The percentage of thymidine replacement was measured by the technique described by Belanger et al. (1987) and Franken et al. (1997a). 2.3. Irradiation Cells were irradiated with single doses up to 4 Gy in case induction of exchange frequencies was studied and up to 8 Gy when clonogenic survival was determined. Irradiations were performed with c-rays from a 137 Cs source at a dose rate of about 0.8 Gy/min. 2.4. Clonogenic assay At 24 h after irradiation the cells were trypsinized and replated in appropriate dilutions in six-well macroplates. Eight days after inoculation in the sixwell macroplates, the colonies were Ž xed and stained in 6% glutaraldehyde with 0.05% crystalviolet. Colonies of 50 cells or more were scored as originating from a single clonogenic cell. The plating eÝ ciency of these cells was 100%. Surviving fractions (S(D)/S(0)) after dose D, corrected for toxicity from IdUrd alone, (S(0)), were calculated and survival curves were analysed using BMDP (Los Angeles, USA) statistical software Ž tting

Correlation between cell reproductive death and chromosome aberrations the data by multiple regression according to the LQ formula: S(D)/S(O) = expÕ

(aD+bD2 ) (Barendsen 1982)

2.5. Metaphase preparation In separate experiments cells were treated to prepare metaphase slides. After irradiation the cells were allowed to repair for 24 h and then subsequently replated from a 75 cm2 to a 162 cm2 culture  ask. Following a further 24 h, colcemid (0.05 mg/ml) was added to the growth medium and mitotic cells were shaken o Ú after 2.5 h. Metaphase spreads were prepared according to standard cytogenetic procedures. Brie y, cells were washed with phosphate-buÚ ered saline (PBS), subsequently treated with hypotonic KCl solution (0.075 m) and Ž xed in methanol/acetic acid (3:1). Finally, the cell suspension was dropped on pre-cleaned slides and air-dried. 2.6. Fluorescence in situ hybridization Metaphase spreads were hybridized to commercially available whole-chromosome-speciŽ c biotinylated probes of chromosome 2 and the X-chromosome (Cambio; Cambridge, UK) using the method described by Pinkel et al. (1986) and Natarajan et al. (1992). Painting of the chromosome was accomplished by applying 100 ml Avidin-Cy3 ( Jackson Labs Inc, Westgrove, PA, USA) in SSCT with 1% BSA. Slides were counterstained with DAPI (2.5 mg/ml) in PBS and embedded in anti-fade solution (Vecta Shield; Vector Laboratories, Burlingame, CA, USA). 2.7. Scoring of aberrations

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metaphases was measured. For this purpose, DAPIstained metaphases and metaphases after FISH were photographed with a CCD camera. After correcting values to the genome DNA content for normal human cells, similar values for DNA contents of the X-chromosome and chromosome 2 were as obtained by Boschman (1992) using  ow cytometry. 3. Results 3.1. EÚects on cell growth and percentage of thymidine replacement by IdUrd The incorporation of IdUrd by itself produced no growth delay or reduction in the plating eÝ ciency. After incubation with 4 mm IdUrd, the percentage of thymidine replacement in the DNA of cells in plateau-phase cultures at the time of irradiation was 4.7Ô 0.1%. 3.2. Radiation dose survival The survival curves obtained for non-IdUrdtreated and IdUrd-treated cells and irradiated in plateau phase are shown in Ž gure 1. Linear–quadratic analysis demonstrated that the value of a in radiosensitized cells increased approximately by a factor of 4; the value of b was not signiŽ cantly changed (table 1). 3.3. Fluorescence in situ hybridization The SW-1573 cells contain between 60 and 67 chromosomes. To study the relationship between the yield of exchanges and radiation doses, the X-chromosome and chromosome 2 of the SW-1573

Slides were examined using a  uorescence microscope (Ortholux II; Leica, Wetzlar, Germany) under green light excitation (552 nm) and emission Ž lters (615 nm) to detect paint and excited with a UV (372 nm) emission Ž lter (456 nm) to detect total DNA (DAPI). A total of 400 to 600 metaphases were scored for each dose and each chromosome. All aberrations involving a painted chromosome and an unpainted chromosome were scored as exchanges (Tucker et al. 1995, Knehr et al. 1998). Terminal and reciprocal exchanges were scored as one event. Interstitial exchanges were scored as two events. Dose–eÚ ect curves for the induction of exchanges by radiation were analysed using the LQ formula: F(D)= aD+bD2 To calculate the relative DNA content of the X-chromosome and chromosome 2, the length of all the chromosomes from photographs of 10 well-spread

Figure 1. Plateau-phase SW-1573 cells plated 24 h after irradiation without IdUrd ( * ) and after incubation with 4 mm IdUrd ( + ). Each point represents the mean value of three diÚ erent experimentsÔ SEM. The curve is a linear– quadratic Ž t to the data.

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Table 1. LQ-parameters describing cell survival and exchanges with and without IdUrd-radiosensitization. LQ parameter a Cell survival X-chromosome Chromosome 2 Total genome realtive to X Total genome relative to 2 a

a (GyÕ

1

) and b (GyÕ

2

a b a b a b a b a b

Control 0.09 Ô 0.046Ô 0.018Ô 0.002Ô 0.031Ô 0.005Ô 0.50 Ô 0.064Ô 0.40 Ô 0.065Ô

0.02 0.002 0.005 0.001 0.009 0.002 0.13 0.027 0.11 0.025

IdUrd-sensitized 0.37 Ô 0.033Ô 0.031Ô 0.009Ô 0.058Ô 0.007Ô 0.86 Ô 0.25 Ô 0.74 Ô 0.09 Ô

0.04 0.006 0.009 0.004 0.006 0.003 0.25 0.11 0.07 0.03

Enhancement factor 4.1 0.7 1.7 3.9 1.9 1.4

).

cells were selected. These chromosomes exhibited no spontaneous exchanges. Of the X-chromosome two copies and of chromosome 2 three copies were present in over 95% of the metaphases studied. According to the chromosome length measurements, the relative lengths of the X-chromosomes and chromosomes 2 were 3.6Ô 0.3% and 7.8Ô 0.6% of the complete genome, respectively. Dose–eÚ ect relationships of yields of exchanges for the X-chromosome and chromosome 2 are presented in Ž gure 2. In these Ž gures, the yield of exchanges with and without radiosensitization with 4 mm IdUrd are shown. Linear–quadratic dose–eÚ ect relationships were obtained for both chromosomes studied with and without IdUrd-radiosensitization. The values of a and b and the derived a- and b-enhancement factors are listed in table 1. The values of a and b for the X-chromosome are lower than the corresponding values for chromosome 2. After incorporation of IdUrd in the DNA for both chromosomes a clear increase of almost a factor of 2 for the value of a was found (table 1). The value of b for the X-chromosome increased by a factor 3.9 and for chromosome 2 an increase of b by a factor 1.4 was observed. Extrapolation of the values of LQ parameters for the single chromosomes to the complete genome resulted, for the X-chromosome, in a and b values of 0.50 GyÕ 1 and 0.064 GyÕ 2 respectively and for chromosome 2 in 0.40 GyÕ 1 and 0.065 GyÕ 2 respectively. Figure 3 shows the overall linear regression analysis between the exchange yield and the surviving fraction. An r-value of 0.96 was obtained for this relationship. 4. Discussion The results show signiŽ cant diÚ erences in the parameters a for cell reproductive death and for chromosomal exchanges. If the assumption is made

Figure 2. Yield of exchanges per cell in the X-chromosome (upper panel) and in chromosome 2 (lower panel) without ( * ) and with ( + ) incorporation of IdUrd. Each point represents the mean value of three diÚ erent experimentsÔ SEM. The curve is a linear–quadratic Ž t to the data.

Correlation between cell reproductive death and chromosome aberrations

Figure 3. Relationship between the surviving fraction and overall exchange yield in SW cells: X-chromosome without ( D ) and with IdUrd ( E ), chromosome 2 without ( ] ) and with IdUrd ( _ ) (r= 0.96).

that all chromosomes are equally sensitive for the induction of exchanges and an extrapolation is made on the basis of the relative DNA content to obtain values for the whole genome, a-values of 0.50 GyÕ 1 and 0.40 GyÕ 1 are derived. Although these values diÚ er by about 20%, a deŽ nite conclusion about a diÚ erence in susceptibility between these chromosomes cannot be derived because of the uncertainties involved in the data. In a study by Wilt et al. (1994), human colon cancer cells in culture were irradiated with a single dose of 8 Gy. FISH analyses of exchanges in chromosomes 1 and 4 showed a signiŽ cantly larger susceptibility of chromosome 1 over chromosome 4 by a factor of 1.5. In contrast, Boei et al. (1997) compared the exchange yields of chromosomes 1 and 4 of human lymphocytes and showed that after a single dose of 2 Gy equal frequencies of colour junctions occurred, although chromosome 1 is a factor 1.3 larger than chromosome 4. The authors concluded that the frequencies of reciprocal exchanges showed a signiŽ cant over-involvement of chromosome 4 over 1, whereas dicentric frequencies were close to the values expected from the DNA content. These diÚ erences could be due to the diÚ erent single doses applied or to the diÚ erence in cell line investigated in these studies. At larger doses the quadratic term plays a larger role. According to the authors’ results, the b-values do not diÚ er signiŽ cantly: 0.064 GyÕ 2 compared to 0.065 GyÕ 2 . Even if radiation-induced chromosome damage did occur at random along DNA, as suggested by Sachs et al. (1992), diÚ erences in aberration yield might be caused by diÚ erences in repair capacity among cell lines and non-randomness in repair among chromosomes. DiÚ erences in aberration yield after single

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doses of radiation have been observed between chromosomes in other tumour cell lines as well as in normal human lymphocytes and Chinese hamster cells (Wilt et al. 1994, Dominguez et al. 1996, Barquinero et al. 1998). The diÚ erences in radiosensitivity between diÚ erent chromosomes have been explained by non-random radiation-induced DNA damage and repair and by interchromosomal diÚ erences in interstitial telomeric DNA sequences (Alvarez et al. 1993, Natarajan et al. 1996). Virsik-Peuckert et al. (1997) measured dicentrics and exchanges in human skin Ž broblasts and in lymphocytes from the same individuals and observed signiŽ cant diÚ erences in the yield as well as in the shapes of the dose responses. Their results suggest that repair processes with diÚ erent eÝ ciencies might be active in cells of diÚ erent types of tissues. Enhancement of radiation damage by incorporation of IdUrd has been demonstrated in the present study’s results for cell reproductive death as well as for exchanges. The increase of a for cell death by a factor of about 4 is larger than for exchanges: 1.7 and 1.9 respectively. It is of interest to point out that these studies were performed with non-cycling cells that are allowed to repair potentially lethal damage (PLD) before the eÚ ects were assessed. This procedure eliminates diÚ erences that might result from variations in PLD repair. Decrease in cell clonogenic capacity due to incorporation of diÚ erent HP has been measured for many cell lines, in particular with cells in plateau phase (Franken et al. 1997a). For the values of b, diÚ erent enhancement ratios could be suggested from table 1. However, it is evident that large uncertainties are involved in the derivation of the value of b. Wilt et al. (1994) also obtained diÚ erences in sensitization of chromosomes 1 and 4 by BrdUrd after a large dose of 8 Gy, where the quadratic term plays a larger part. DiÚ erent incorporation levels of HP in the chromosomes might be responsible for these diÚ erences. The variations in exchange yields obtained for the sensitized X-chromosome are larger than those obtained for chromosome 2 (Ž gure 2). This may be due to the diÚ erence in DNA content between the two chromosomes. The two X-chromosomes contain about two times less DNA than the three chromosome 2. The values of a for exchanges in the total genome shown in table 1, 0.50 GyÕ 1 and 0.40 GyÕ 1 respectively, are evidently much larger than the value of a= 0.09 GyÕ 1 for cell reproductive death. The results suggests that a large majority of the observed exchanges do not cause cell reproductive death. It is well known that radiation damage to chromosomes

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can be detected in subsequent generations of cells after irradiation. It would be of great interest if FISH could be used to measure chromosome aberrations as a predictor of radiosensitivity in human tumour cells. The LQ analyses presented do not suggest a direct correlation of exchanges assessed by FISH with radiosensitivity for cell reproductive death. However, with surviving fraction plotted against overall exchange yield in the X-chromosome and chromosome 2, a very signiŽ cant correlation was obtained (r= 0.96). This is in agreement with results obtained by Coco-Martin et al. (1994). They further suggested that a better correlation could be obtained if survival is compared with the diÚ erences in aberration yield between day 1 and day 14 after irradiation. The rationale is that aberrations still present at 14 days after treatment are evidently not lethal. In conclusion, it is suggested that analysis of dose– response relationships for a suÝ ciently wide range of doses, using the LQ model, provides insight into the correlation of cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and the sensitization by halogenated pyrimidines. Further studies with other cell lines are required to evaluate the generality of these relationships. Acknowledgements This work was supported by the Interuniversity Research Institute of Radiopathology Radiation Protection, J. A. Cohen Institute (IRS project #: 7.1.7.). The Maurits and Anna de Kock foundation is acknowledged for providing the CCD camera. Mrs G. Bakvis is acknowledged for typing the manuscript. References Alvarez, L., Evans, J. W. and Wilks, R., 1993, Chromosomal

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