Mutation Research, 284 (1992) 223-231 223 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

MUT 05180

Chronic 7-irradiation results in increased cell killing and chromosomal aberration with specific breakpoints in fibroblast cell

strains derived from non-Hodgkin's lymphoma patients

Manjula Waghray, David Sigut, Michael Einspenner, Mohammed Kunhi, Sultan T. A1-Sedairy and Mohammed A. Hannan a

Departments of ~ Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia

(Received 7 April 1992) (Revision received 2 June 1992)

(Accepted 23 June 1992)

Keywords: Non-Hodgkin's lymphoma; 3,-Irradiation; Chromosome break

Summary

Cultured skin fibroblast ceils from 6 patients with non-Hodgkin's lymphoma (NHL) and2 clinically normal subjects were compared for cell survival and chromosomal aberration after chronic 7-irradiation. Fibroblasts from an ataxia telangiectasia (AT) homozygote and an AT heterozygote were used as positive controls. Following irradiation, fibroblasts from all 6 NHL patients showed an increase in both cell death and chromosomal aberration (breaks and rearrangements) compared to the normal subjects. The difference in the frequency of chromosomal aberration between the normals and the NHL patients remained virtually unchanged over a period of 24-72 h post irradiation incubation of the ceils. Cell cycle analysis by flow cytometry carried out in 1 normal and 1 NHL fibroblast cell strain showed that more ceils representing the NHL patient were in G2/M phase compared to the normal at various times of cytogenetic analysis. While the AT homozygote appeared to be the most radiosensitive, the AT heterozygote showed a slightly higher incidence of cell death and chromosomal aberration than the normals. The cellular and chromosomal radiosensitivity of fibroblast cell lines from the NHL patients differed slightly from that of the AT heterozygote but clearly occupied an intermediate position between the AT homozygote and the normal subjects. Cells from 3 of the NHL patients showed radiation-induced specific chromosomal breaks involving chromosomes 1, 2, 6, 8, 10 and 11 which correspond to known fragile sites. Such breakpoints associated with increased radiosensitivity may be indicative of predisposi- tion to malignancy in the patients studied.

Correspondence: Dr. M.A. Hannan, Department of Biological Research, King Faisal Specialist Hospital and Research Cen- tre, P.O. Box 3354, Riyadh 11211, Saudi Arabia.

Studies on inherited disorders like ataxia telangiectasia (AT) and xeroderma pigmentosum (XP) have suggested a possible link between DNA repair defects and incidence of cancer (Setlow, 1978; Lehman, 1982). Several investigators have

224

tested this hypothesis by analysing in vitro car- cinogen sensitivity of normal body cells from can- cer patients as well as cancer-prone individuals including AT heterozygotes (Auerbach and Wol- man, 1976; Weichselbaum et al., 1978; Arlett and Harcourt, 1980; Kutlaca et al., 1984; Paterson et al., 1985; Hsu et al., 1989; Little et al., 1989). A number of these studies demonstrated enhanced (cellular) radiosensitivity in certain cancer pa- tients and individuals predisposed to malignan- cies although some failed to confirm these find- ings (Kassakowska et al., 1982; Wang et al., 1986; Little et al., 1987, 1989). Increased radiosensitiv- ity has also been reported in several neurodegen- erative diseases without any known association with neoplasia (Robbins et aI., 1985). In most cases, however, neither the mechanisms underly- ing radiosensitivity nor its genetic consequences (chromosomal alterations and mutagenesis) have been fully investigated.

In earlier studies, we observed that most of the skin fibroblast cell strains derived from Saudi patients with non-Hodgkin's lymphoma (NHL) showed increased sensitivity to chronic y-irradia- tion (Hannah et al., 1989, 1991) indicating that radiosensitivity may be commonly associated with these patients. In the present study, fibroblast cell strains from 6 NHL patients showing in- creased radiosensitivity and 2 normal subjects were analysed for chromosomal aberrations after chronic irradiation. Data showed that enhanced chromosomal breaks/cel l occurred in all of the radiosensitive cell strains derived from the NHL patients, some exhibiting damage at specific chro- mosomal regions corresponding to known fragile sites.

these cell strains, found to show increased sensi- tivity to (chronic) y-irradiation compared to the cell strains from several healthy subjects, were further analysed for radiation-induced chromoso- mal aberrations. In addition, cell lines from 2 normal subjects, 1 AT homozygote and 1 AT heterozygote (isolated by the same procedure) were used to compare the radiation-induced cell killing and chromosomal breakage with those from the NHL patients.

Irradiation and suruival assay Early passage (4-10) cells were grown in Ham's

F12 medium containing the same supplements as in MEM. Cells from the exponential growth phase were harvested, plated in the growth medium (F12) in 100-mm tissue culture plates and grown to confluence. The confluent cultures were then irradiated in a CO 2 incubator, using a medical 6°Co y-source (International Neutronics, Inc., Paio Alto, CA) at a dose rate of 0.0076 Gy /min for 2.5-30 h. After irradiation, the growth medi- um was renewed and the plates were left in the CO e incubator overnight. Cells, at different radi- ation dose points, were then trypsinised and seeded at appropriate densities together with a feeder layer consisting of y-radiation (50 Gy)-in- activated human fibroblasts (60,000 cells/plate). Cells were further incubated, with a weekly medium change, for up to 3 weeks. Macroscopic colonies (> 50 cells/colony) were then washed with phosphate-buffered saline, stained with crys- tal violet and scored as survivors. A plot of per- cent survival, at different radiation doses, was generated for each cell strain using the respective unirradiated survivors as controls.

Materials and methods

Cell strains Skin fibroblast cell strains were developed from

cutaneous biopsies obtained from Saudi NHL patients by growing the skin explants in minimal essential medium (MEM) supplemented with Earle's salts, penicillin (100 U/ml) , streptomycin (100 /zg/ml), glutamine (2 mM) and 10% foetal bovine serum in 25-cm 2 tissue culture flasks. The flasks were incubated at 37°C in a humidified (80%) atmosphere with 5% CO 2, 95% air. Six of

Cytogenetic assay Following overnight post-irradiation incuba-

tion the cells from different dose points were trypsinised and transferred to fresh medium in tissue culture dishes. After further incubation for different periods of time, colcemid was added to the cell cultures at a concentration of 0.05 mg/ml for 1 h. The cells were then harvested by trypsini- sation, exposed to 0.075 M hypotonic KCI solu- tion at 37°C for 20 rain and fixed in cold 1:3 acetic ac id /methanol fixative for 1 h. The cells in suspension were dropped on pre-cleaned, chilled

225

TABLE 1

C H R O N I C R A D I A T I O N D10 VALUES INDICATING DIFFERENCES IN RADIOSENSITIVITY OF FIBROBLAST CELL STRAINS F R O M N O R M A L SUBJECTS, NHL PATIENTS, AT H O M O Z Y G O T E AND AT H E T E R O Z Y G O T E

Cell strain a Clinical status Age Sex Passage Number of Di0 value of donor (years) No. experiments (cGy)

C128552 b Normal subject 29 F 3-18 5 700-930 C247236 Normal subject 67 F 4-13 3 660-880 C243657 Normal subject 17 M 4-10 3 620-715 C148805 Normal subject 48 M 5-11 2 620-680 C241963 b Normal subject 26 F 7 1 620

L234794 b NHL patient 71 M 6-10 2 420-475 c L213298 b NHL patient 36 M 4 - 6 2 220-460 c L213725 b NHL patient 59 M 5 - 8 2 400-470 c L244455 b NHL patient 71 M 4-10 3 400-540 c L266468 b NHL patient 90 M 7 1 500 L261634 b NHL patient 63 M 10 1 480

AT209890 b AT homozygote 9 M 7 - 9 2 140-145 AT172600 b A T heterozygote 26 F 5 - 7 3 340-430

" All cell strains were developed from skin biopsies taken before the onset of any therapy. b Used in cytogenetic studies.

c Chronic radiosensitivity for cell killing of these 4 cell strains was reported earlier (Hannah et al., 1991) and the remaining 2 are new isolates.

TABLE 2

F R E Q U E N C I E S OF S P O N T A N E O U S AND C H R O N I C R A D I A T I O N - I N D U C E D C H R O M O S O M A L A B E R R A T I O N IN CELL STRAINS D E R I V E D F R O M NHL PATIENTS, AT H O M O Z Y G O T E , AT H E T E R O Z Y G O T E AND H E A L T H Y SUBJECT

Fibroblast Clinical Chromosal aberra t ions/100 cells cell strain status of

Dose Breaks Trans loca t ions / R i n g s / Average donor

(Gy) Inversions Dicentrics breaks/cel l

209890 AT 0.0 20 9 1 0.40 homozygote 1.8 44 34 4 1.20

4.6 176 172 15 5.50

172600 AT 0.0 5 1 0 0.07 heterozygote 1.8 12 7 0 0.26

4.6 22 18 2 0.62

L234794 N H L 0.0 6 1 0 0.08 patient 1.8 20 8 1 0.38

4.6 50 30 4 1.18

L213725 NHL 0.0 7 3 0 0. t 3 patient 1.8 30 12 2 0.58

4.6 48 48 6 1.56

L213298 NHL 0.0 6 2 0 0.10 patient 1.8 26 15 3 0.62

4.6 58 55 8 1.84

C128552 Normal 0.0 5 0 0 0.05 subject 1.8 8 1 0 0.10

4.6 18 3 0 0.24

226

slides and the chromosomes were trypsin-Giemsa banded. Chromosome analysis in each cell strain was performed on 100 (coded) banded meta- phases for each dose point. Breaks and rear- rangements (i.e., rings, translocations, inversions, dicentrics) were scored (as chromosomal aberra- tions) and the karyotypic analysis was performed as defined by ISCN (1985) nomenclature. Rear- rangements were assessed as two breaks per cell. The baseline chromosome breakage for each sample was calculated from the respective un- treated cultures.

Cell cycle (DNA content) analysis Cultured cells were processed using Cycletest

reagents (Becton Dickinson, Erembodegem, Bel- gium).

Briefly, the cells (approximately 5 x 105) were centrifuged at 300 x g for 5 min. The culture medium was then aspirated and the cells resus- pended in 0.2 ml of the citrate buffer. Next, 1.8 ml of solution A (trypsin) was added and the tube was gently inverted for 10 min. 1.5 ml of solution B (trypsin inhibitor) was added next and the tube was inverted as before for another 10 min. Fi- nally, 1.5 ml of solution C (propidium iodide) was added and again the tubes were inverted for 10 min (in the dark). The samples were then filtered through a 30-/xm nylon mesh and immediately analysed on the FACScan flow cytometer (Becton Dickinson, San Jose, CA). The FACScan was equipped with a DDM (doublet discrimination module) and CelIFIT software.

Results and discussion

An increased sensitivity of fibroblast cell strains from NHL patients to chronic y-irradiation has already been reported (Hannah et al., 1989, 1991). Further experiments were carried out in the pre- sent study with fibroblast strains from the NHL patients (4 reported for radiosensitivity earlier and 2 new isolates) to demonstrate an association of cellular and chromosomal radiosensitivity. Fig. 1 illustrates the survival curves obtained after chronic irradiation of cell strains from 2 NHL patients compared to those from 1 AT homozy- gote and 1 AT heterozygote and 5 normal sub- jects including the 2 cases used in cytogenetic

11111-

10"

m 1.0" E

0.1'

L213725 O 1.213298 •

AT209890 A ATH 172600 •

N O R M A L RANGE

I I I I I I 2 4 6 8 10 12

CHRONIC GAMMA RADIATION ( DOSE iN GY) Fig. 1. Cell survival curves of fibroblasts from an AT homozy- gote, an AT heterozygote, 2 NHL patients compared to those

obtained from 5 healthy subjects providing a normal range.

analysis. For simplicity of presentation, survival curves for all of the cell strains examined are not shown but their D~0 values (radiation doses re- sulting in 10% survival) are given in Table 1. These data showed that the survival response to irradiation of the cell strains varied with experi- ments and the test subjects serving as donors. The problem of intrinsic variation in radiosensi- tivity of normal subjects was discussed in earlier reports (Little et al., 1988; Hannan et al., 1991) and taken into consideration in evaluating the relative radiosensitivity of various cell strains in the present study. Despite the inter-strain varia- tion in sensitivity to radiation, the cell survival curves of the 2 control cell lines (healthy subjects) used for cytogenetic investigation in the present study fell within the range representing normal subjects while the cell strains from NHL patients, as a group, consistently appeared to be more radiosensitive. In fact, the data presented in Fig.

1 and Table 1 showed that the radiation response of the cell strains from NHL patients occupied an intermediate position between an AT homozy- gote and the normal subjects, and only slightly differed from that of the AT heterozygote. Al- though an AT heterozygote-like response was observed in NHL fibroblasts, it is not possible to know whether or not these NHL patients were in fact carriers of the gene for AT since no AT gene probe is available at present nor is there definite evidence of the occurrence of AT homozygotes in their families. The mechanisms underlying the increased radiosensitivity of the fibroblast cell strains from the NHL patients are not known. Studies on several inherited disorders led to the suggestion that defective DNA repair processes may be responsible for the relationship between increased radiosensitivity and predisposition to malignancies (Cleaver, 1970; Setlow, 1978; Pater- son et al., 1981; Lehman, 1982; Bohr and Kober, 1985). The genetic basis of increased susceptibil- ity to cancers has been indicated by the associa- tion of enhanced carcinogen sensitivity for cell

227

killing with increased mutagenesis and/or chro- mosomal aberrations in the cell strains derived from AT and XP patients and fibroblasts from breast cancer patients (Taylor, 1982; Bigbee et al., 1989; Tachibana et al., 1989; Nagasawa et al., 1990). An implication of possible DNA repair defects with cancer should be supported by fur- ther evidence of increased genetic/chromosomal alterations in cell strains from various cancer- prone disorders as well as cancer patients show- ing increased cellular sensitivity to carcinogens.

In the present study, we examined the inci- dence of chromosomal aberrations (breaks and rearrangements) in the radiosensitive cell strains from 6 NHL patients in comparison with 2 nor- mal subjects as well as 1 AT homozygote and 1 AT heterozygote. First, we studied the chromoso- mal aberrations in unirradiated and irradiated (1.8 and 4.6 Gy) cells from 3 NHL patients and 1 normal subject, an AT homozygote and an AT heterozygote, all harvested after a 48-h post- irradiation growth in fresh medium. The data presented in Table 2 show that the incidence of

TABLE 3

F R E Q U E N C I E S OF S P O N T A N E O U S AND C H R O N I C R A D I A T I O N - I N D U C E D C H R O M O S O M E A B E R R A T I O N S IN FIBROBLAST CELL STRAINS D E R I V E D F R O M 3 NHL PATIENTS A N D 2 N O R M A L SUBJECTS A F T E R D I F F E R E N T PERIODS OF P O S T - I R R A D I A T I O N INC UB AT ION

Fibroblast Clinical Chromosomal aberra t ions/100 cells cell strain status of

Dose Breaks Trans loca t ions / R i n g s / Average donor (Gy) Inversions Dicentrics breaks/cel l

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

L266468 NH L 0 3 4 2 1 1 1 0 0 0 0.10 0.12 0.08 patient 1 11 8 10 6 6 5 1 1 0 0.50 0.44 0.40

3.5 20 22 21 20 19 20 2 3 2 1.28 1.32 1.30

L261634 NHL 0 3 4 2 0 0 0 0 0 0 0.06 0.08 0.04 patient 1 7 4 5 4 6 5 0 1 0 0.30 0.38 0.32

3.5 14 13 16 10 14 12 2 2 1 0.76 0.90 0.84

L244455 NHL 0 2 3 3 1 0 0 0 0 0 0.08 0.06 0.04 patient 1 8 11 9 4 4 6 1 1 1 0.36 0.42 0.44

3.5 16 21 19 16 14 16 3 3 2 1.08 1.18 1.10

C128552 Normal 0 2 1 3 0 0 0 0 0 0 0.04 0.02 0.06 subject 1 5 7 4 0 1 1 0 0 0 0.10 0.18 0.12

3.5 10 9 11 2 3 3 0 0 0 0.28 0.30 0.34

C241963 Normal 0 3 2 2 0 0 0 0 0 0 0.06 0.04 0.04 subject 1 6 8 7 1 1 1 0 0 0 0.16 0.20 0.18

3.5 12 10 9 2 4 3 0 0 0 0.32 0.36 0.30

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chromosomal aberrations was the highest in the AT homozygote and the lowest in the normal subject. However, relative to the normal subject, the cell strains from the NHL patients showed increased chromosomal breaks/cell . The AT het- erozygote also showed increased chromosomal aberrations/cell compared to the normal but its chromosomal radiosensitivity was less than that of the NHL patients. These data, thus, demon- strate the increased chromosomal aberrations as- sociated with intermediate cellular sensitivity to chronic irradiation in cell strains isolated from the NHL patients.

The increased frequency of cells with chromo- somal aberrations observed in patient cell strains

could be due to more mitotic activity in the normal cells resulting in the loss of cells carrying abnormal chromosomes. In order to rule out this possibility we carried out cytogenetic studies in cell strains from 3 NHL patients and 2 normals at different time points (24, 48 and 72 h) after post-irradiation growth in fresh medium. The data presented in Table 3 show that there was no notable change in the frequency of cells with chromosomal aberrations in different cell strains studied over these time periods. However, the cell lines from the NHL patients consistently showed an increased incidence of chromosomal breaks/cel l compared to their normal counter- parts. We also examined the DNA content of 1

Fig. 2. (a) Cell cycle distributions of control fibroblast cells. From front to back: 0, 24, 48, 72 h post plating (no irradiation). The X-axis gives DNA content (propidium iodide fluorescence). The Y-axis gives cell counts, normalised. The first peak in each distribution represents cells in G 0 / G l phase, the second peak cells in G z / M phase. Counts in between are cells in S phase. G 0 / G t doublets have been excluded by electronic gating. (b) Cell cycle of fibroblast cells from NHL patients, 0 -72 h post plating. Same conditions as in a. (c) Cell cycle distributions of control cells 0, 24, 48, 72 h post irradiation. Cells were re-plated after irradiation (3.5 Gy). Other details as in a. (d) Cell cycle of fibroblast cells from NHL patients, 0 -72 h post irradiation (3.5 Gy).

Again, cells were re-plated after irradiation. Other details as in a.

normal cell strain (C128552) and 1 patient's cell strain (L266468) by flow cytometry at the differ- ent times of cytogenetic analyses. Fig. 2 (a-d) shows a set of representative DNA content curves for the normal and the NHL cell strains before and after irradiation (3.5 Gy). Surprisingly these data showed that more ceils were in G2/M phase in the case of fibroblasts from the NHL patient than those from the normal subject at various times of chromosomal studies. While these re- sults suggested a possible aberration in the cell cycle pattern of the cell strain derived from the NHL patient, they showed that the higher fre- quency of chromosomal aberration in the patient's cells was not due to a lack of cell proliferation in them. Unfortunately, these data did not provide information regarding the number of cell cycles undergone by each cell showing chromosomal aberration, and are, thus, of limited value in interpreting the role of cell divisions in eliminat- ing the progeny carrying abnormal chromosomes. Further studies are needed to examine the cell cycle characteristics of the fibroblasts from NHL patients to find whether a defect in the cell cycle could be responsible for their increased cellular and chromosomal radiosensitivity.

A careful examination of the karyotypes in cell strain from 3 NHL patients after irradiation with 1 or 1.8 Gy led to the identification of breaks in

229

specific chromosomal regions while none of these were found in the normals (Table 4). These chro- mosomal regions correspond to known fragile sites in the neighbourhood of which there exist various proto-oncogenes (Heim and Mitelman, 1987; Hecht, 1988). These chromosomal breakpoints observed in the irradiated fibroblasts of the NHL patients are very interesting indeed, as they are known to be involved in both lymphomas and leukemias (Yunis, 1984; Fifth International Workshop on Chromosomes in Leukemia-Lym- phoma, 1987; Le Beau, 1990). However, the sig- nificance of this observation remains intriguing as the radiation-induced breakpoints we have ob- served are in the cultured fibroblasts from NHL patients while chromosomal translocations involv- ing these breakpoint hotspots have been reported in the cancer cells. Also, the data on the expres- sion of fragile sites after chronic irradiation need to be collected in a manner permitting extensive statistical analysis for every specific breakpoint hotspot to determine the significance of occur- rence. In the present data, Fisher's exact test was used to compare the incidence of specific break- points in NHL fibroblasts with zero incidence in normals and at least two breakpoints showed significant results (P = 0.03). Further studies us- ing more cell strains from NHL patients and quantitating their chromosomal breakpoint

TABLE 4

OCCURRENCE OF SPECIFIC CHROMOSOME BREAKS CORRESPONDING TO FRAGILE SITES FOLLOWING CHRONIC y-IRRADIATION IN FIBROBLAST CELL STRAINS DERIVED FROM 3 NHL PATIENTS (PER 100 METHAPHASE CELLS ANALYSED IN EACH)

Fibroblast Radiation Chromosomal Frequency Oncogenes known cell strain dose location of (% of cells to be located at or

(Gy) breakpoints showing the near these sites specific breakpoints)

L213725 1.8 2p 13 2 (0.1) N-myc 8q22 2 (0.1) C-mos, C-myc 6q21 1 (0.3) myb

L266468 1.0 10q21 2 (0.1) 8q24 3 (0.03) * C-myc

L261634 1.0 lp32 3 (0.03) * Blym-l, L-myc 11q14 1 (0.3) int-2, ets-I

bcl-1

Figures in parentheses are P values obtained by Fisher's exact test. * Significant.

230

hotspots compared to those in a group of healthy subjects and asymptomatic relatives of the pa- tients may lead to an understanding of the possi- ble role of the chronic radiation-induced specific breakpoints in cancer predisposition in radiosen- sitive populations. Also, one may expect cluster- ing of NHLs or other malignancies among rela- tives of the carcinogen-sensitive patients showing expression of fragile sites. Although none of the 6 NHL patients in this study showed a family his- tory of cancer this subject is at present under further investigation using extensive question- naires for all patients examined for increased radiosensitivity.

The usefulness of chronic irradiation in detect- ing increased chromosomal aberration in AT het- erozygotes was reported by us earlier (Waghray et al., 1990). The present studies further demon- strated the advantage of chronic irradiation in not only detecting the enhanced cellular and chromosomal radiosensitivity of NHL patients but also identifying chromosomal fragile sites in the fibroblasts from these patients. Whether these phenomena occur after chronic irradiation of cell strains from other cancer patients or cancer-prone disorders will be the subject of further studies.

Acknowledgements

The authors acknowledge the support from the King Faisal Specialist Hospital and Research Centre through Project No. 85-0009. Also, they thank Dr. Bashir A. Khan of the Department of Biostatistics and Scientific Computing for the sta- tistical analysis.

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