Carcinogen-induced chromosome breakage in chromosome instability syndromes

Carcinogen-induced Chromosome Breakage in Chromosome Instability Syndromes Arleen D. Auerbach and Sandra R. Wolman

ABSTRACT: Cultured fibroblasts from patients with chromosome instability syndromes (Fanconi's anemia (FA), ataxia telangiectasia (AT), Bloom's syndrome (BS), xeroderma pigmentosum (XP)) and from normal individuals were examined for their susceptibility to diepoxybutane (DEB)-induced chromosome breakage. Clear differences in patterns of spontaneous and induced chromosome breakage in the different syndromes were found. In two of the syndromes (FA and AT), chronic exposure to a low concentration of the carcinogen induced extensive chromosome damage without reduction in cell viability. Exposure to the same concentration of carcinogen had no clastogenic effect on BS, XP, or normal fibroblasts. Furthermore, when normal fibroblasts were exposed to a higher dose of the same chemical, there was significant reduction in viability with little increase in chromosome aberrations. These experiments separate the clastogenic effect of the carcinogen from its cytotoxic effect, and show genetic differences in susceptibility of cells to carcinogen-induced chromosome damage. INTRODUCTION The current focus on environmental causes of cancer has p a i d insufficient attention to the differing susceptibilities of the exposed p o p u l a t i o n s . I n d i v i d u a l s affected with the autosomal recessive s y n d r o m e s F a n c o n i ' s a n e m i a (FA), ataxia telangiectasia CAT), Bloom's s y n d r o m e (BS), and xeroderma p i g m e n t o s u m (XP) have an increased p r e d i s p o s i t i o n to cancer [1]. In three of these s y n d r o m e s (FA, AT, XP) there is evidence for defective repair of damage to DNA by environmental agents [2]. Cultured cells from i n d i v i d u a l s with FA, AT, and BS exhibit spontaneous c h r o m o s o m e instability, w h i l e cells from patients w i t h FA, AT, and XP have increased susceptibility to the i n d u c t i o n of c h r o m o s o m e breakage by chemical or p h y s i c a l carcinogenic agents [3 - 8]. A l t h o u g h observation of c h r o m o s o m e aberrations p r o v i d e d the original spur to the somatic m u t a t i o n theory of cancer [9], their role in the etiology of m a l i g n a n c y has long been disputed. The correlations between mutagenicity and carcinogenicity of chemical carcinogens [10,11] p r o v i d e evidence that DNA damage is i m p o r t a n t in carc i n o g e n - i n d u c e d neoplastic transformation. Gross chromosomal abnormalities are one form of heritable change, and recent demonstrations of specificity of chromosome change in tumors [12] have r e a w a k e n e d interest in c h r o m o s o m e damage as a possible route to carcinogenesis. The association of c h r o m o s o m e instability with carcinogenesis in the autosomal recessive s y n d r o m e s is further evidence for an associaFrom the Laboratoryof Genetics,Memorial Sloan-KetteringCancer Center, New York, New York, and the Department of Pathology, New York University School of Medicine, New York, New York. This paper is part of a doctoral thesis submitted to New York University, 1977 (by A.D.A.}. Address reprint requests to: Dr. Arleen D. Auerbach, Laboratory of Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Received January 18, 1979; accepted March 1, 1979. © Elsevier North Holland, Inc., 1979 Cancer Genetics and Cytogenetics1, 21-28 (1979) 4165-4608/79/01002108502.25

22

A.D. Auerbach and S. R. Wolman tion between mutagenesis and carcinogenesis. Cells cultured from patients with these syndromes can provide a model for the study of the interaction of genetic susceptibility with carcinogenic environmental agents. We have studied the susceptibility of cultured fibroblasts from patients with each of the four chromosome instability syndromes, and suitable controls, to further chromosome damage by diepoxybutane (DEB), a direct-acting mutagen and carcinogen [13-16]. If such damage is mutational in the sense that it is heritable, it must not be toxic to the altered cell. Therefore, in our studies we have examined the effects of the carcinogen on fibroblasts at exposures where no significant level of toxicity was detected.

MATERIALS AND METHODS Cell Cultures Fibroblast cultures derived from patients with FA (C12, C17) and AT (C27, C45) were obtained from Dr. M. Swift, University of North Carolina. XP fibroblasts (CRL 1158, complementation group C) were obtained from the American Type Culture Collection. BS fibroblasts (HG916) were obtained from Dr. J. German, The New York Blood Center. Controls were normal fihroblasts derived from newborn foreskins (NSF-2, NSF-3), obtained from Dr. R. Cox, New York University School of Medicine, normal fibroblasts from adult skin biopsies (C39, C40), obtained from Dr. M. Swift, and trisomy 18 fibroblasts (755), derived from a skin biopsy in our laboratory. All studies were done on cells between the 8th and 18th passage. Cells were cultured in Dulbecco's modified Eagle medium supplemented with 10 or 15% fetal bovine serum, L-glutamine (2 mM), penicillin (50 units/ml), and streptomycin (50 ~g/ml) (Grand Island Biological Co, GIBCO). Cultures were incubated in a 10% CO 2 atmosphere at 37°C. DEB was kindly provided by Dr. B. M. Goldschmidt (New York University School of Medicine). At the time of use the pure chemical, a freshly distilled liquid, was diluted to an appropriate concentration with phosphatebuffered saline (PBS) and then added to the medium.

Toxicity Studies Fibroblasts were plated in 25 cm 2 Falcon tissue culture flasks at a density of 3 × 105 cells/flask. Twenty-four hours after subculture, cells were exposed to various concentrations of DEB. Final concentrations of DEB in the culture medium were 1.0, O.1, or 0.01/~g/ml. Four days after plating (DEB has a half-life of 4 days in aqueous solution) cultures were washed with PBS and fed with medium to which fresh carcinogen in appropriate concentrations was added. Cells thus received a chronic exposure to DEB over a 6-day period. Cell counts and dye exclusion studies using trypan blue were done over the 7-day period on chemically treated cells and on untreated controis. All counts were done in triplicate.

Chromosome Studies Fibroblast cultures received a chronic exposure to DEB (0.01/~g/ml) over a 6-day period, while replicate cultures of each cell strain served as untreated controls. This concentration of DEB had little effect on the growth rate and viability of treated cells, enabling us to separate the chromosome-damaging effect of the chemical from its cytotoxic effect. The cells were harvested for chromosome studies after replating and growth for 48 hr in carcinogen-free medium in order to study the residual effect of DEB on the surviving and dividing population of cells. Slides were stained with ace-

23

Carcinogen-induced Chromosome Breakage

to-orcein and metaphase figures were scored for chromosome number and for the presence of chromosome abnormalities. Chromatid and isochromatid gaps, breaks, and rearrangement figures were recorded. An achromatic area more than a chromatid in width was scored as a break. Gaps were not included in calculations of breakage rates. Dicentrics, quadriradials, and translocation figures were scored as rearrangements, and each was counted as two breaks in the calculation of breakage frequencies. RESULTS

Normal Fibroblasts Several strains of normal fibroblasts with differing growth rates were examined. Figure I shows the growth rate of DEB-treated and untreated NSF-3 and C40 cells. Untreated NSF-3 cells underwent approximately three cell doublings in 7 days, while C40 cells had approximately two doublings. A concentration of 0.01 pg/ml of DEB was nontoxic in each of these cell strains, while a lO-fold increase in DEB concentration, to 0.1 ftg/ml, caused a marked reduction in the 7-day growth achieved by NSF-3

Figure 1 Effectof DEB on growth of normal fibroblasts. DEB at a concen~ation of 0.01 pg/ml did not affect cell growth, while 0.1 ~g/ml caused a marked reduction in the growth achieved by NSF-3 in 7 days. (Q) NSF-3 untreated; (O) NSF-3 treated with DEB (0.01/~g/ml); (~) NSF-3 treated with DEB (0.1 /tg/ml); ill) C40 untreated; (D) C40 treated with DEB (0.01 /~g/ml).

0 12 U

"6 Z

9

I

2

3

4

TIME (Days)

5

6

7

24

A . D . A u e r b a c h a n d S. R. W o l m a n

cells. N o g r o w t h o c c u r r e d at 1 . 0 / ~ g / m l . T h e r e w e r e also n o d i f f e r e n c e s i n g r o w t h bet w e e n N S F - 2 c e l l s t r e a t e d w i t h 0 . 0 1 / ~ g / m l DEB a n d u n t r e a t e d ceils, w h i l e 0.1 ~ g / m l DEB c a u s e d a 5 0 % r e d u c t i o n i n t h e g r o w t h r a t e ( n o t s h o w n } . E a c h of t h e n o r m a l s t r a i n s s t u d i e d c y t o g e n e t i c a l l y (NSF-2, NSF-3, C39, C40) h a d a c h r o m o s o m e c o m p l e m e n t of 46,XY. T h e effect of DEB t r e a t m e n t o n c h r o m o s o m e b r e a k a g e i n t h e s e c e l l s is s h o w n i n T a b l e 1. T r e a t m e n t w i t h a n o n t o x i c c o n c e n t r a t i o n of 0.01 ~ g / m l DEB h a d n o d e t e c t a b l e effect o n c h r o m o s o m e b r e a k a g e , e v e n i n t h e s l o w e r - g r o w i n g s t r a i n C40, w h i c h h a d a r e l a t i v e l y h i g h r a t e of s p o n t a n e o u s c h r o m o s o m e b r e a k a g e , It is i m p o r t a n t to n o t e t h a t t r e a t m e n t of N S F - 3 c e l l s w i t h 0 . 1 / ~ g / m l DEB, a c o n c e n t r a tion which caused a marked decrease in the overall growth achieved in 7 days, n e v e r t h e l e s s f a i l e d to i n d u c e a s i g n i f i c a n t i n c r e a s e i n c h r o m o s o m e b r e a k a g e .

Ataxia Telangiectasia Fibroblasts Cell s t r a i n s f r o m t w o A T p a t i e n t s w e r e s t u d i e d (C27,C45). T r e a t m e n t w i t h 0 . 0 1 / x g / m l DEB c a u s e d n o s i g n i f i c a n t r e d u c t i o n i n t h e 7 - d a y g r o w t h rate of t h e s e cells. B o t h A T cell s t r a i n s h a d a c h r o m o s o m e c o m p l e m e n t of 46,XY. Cell s t r a i n C27 h a d a b o u t t w i c e t h e s p o n t a n e o u s b r e a k a g e of s t r a i n C45 ( T a b l e 1). W i t h i n e a c h cell s t r a i n , t h e a m o u n t

Table 1

DEB-induced chromosome breakage in normal, ataxia telangiectasia, and Bloom's syndrome fibroblasts

Cell strain

No. of No. of Passage cells Chromatid Chromosome Fragments + Rearrange- breaks n u m b e r examined breaks breaks deletions ments a per cell

Normal NSF-3 untreated 10 0.01/~g/ml DEBb 10 0.1 ~g/ml DEB 10 NSF-2 untreated 17, 18 0.01/zg/ml DEB 18 C39 untreated 11 0.01 ~g/ml DEB 11 C40 untreated 12 0.01/~g/ml DEB 12 Ataxia telangiectasia C27 untreated 10 0.01/~g/ml DEB 10 C45 untreated 12 0.01 p.g/ml DEB 12 Bloom's syndrome HG916 untreated 17 0.01 ~g/ml DEB 17

100 100 100

2 4 6

0 0 1

0 0 1

0 0 0

0.02 0.04 0.08

100 100

0 0

0 0

0 0

0 0

0 0

100 100

2 2

0 0

1 3

0 0

0.03 0.05

100 100

2 5

2 0

2 0

1 1

0.08 0.07

50 50

7 16

1 3

3 2

0 1

0.22 0.46 c

100 100

6 7

0 1

3 4

2 7

0.13 0.26 c

100 100

5 5

0 2

9 9

13 10

0.40 0.36

aRearrangements include translocations and dicentrics and are scored as 2 breaks. bCells were exposed to DEB for 6 days and subcultured in medium without DEB. Chromosome preparations were made 48 hr after subculture. "Differences between DEB-treated and untreated replicate cultures were significant (P<0.05).

Carcinogen-induced Chromosome Breakage

25

of chromosome breakage doubled after DEB treatment. The difference in breakage between the DEB-treated and the untreated cells was statistically significant (P < 0.05) in each case.

Bloom's Syndrome Fibroblasts One strain of BS fibroblasts (HG916) was studied for susceptibility to DEB-induced chromosome breakage. These cells were examined for sister chromatid exchange (SCE) by Dr. J. German (The New York Blood Center) and showed the characteristically high frequency of exchanges for BS cells [17]. There was no reduction in the growth of DEB-treated cells compared to untreated cells. The cells generally had a numerically normal chromosome complement of 46,XX. However, an unusually large number of metaphases contained stable chromosomal rearrangements which were not clonal but involved a variety of chromosomes in different cells. To determine whether the frequency of cells with morphologically aberrant chromosomes was affected by DEB treatment, karyotypes were prepared from 23 untreated and 23 DEB-treated cells. Fourteen untreated and 13 DEB-treated cells had one or more abnormal chromosomes with additional, missing, or rearranged chromosome segments, indicating no effect of treatment. The overall frequency of chromosomal aberrations in BS fibroblasts is shown in Table 1. There was no increase in either achromatic lesions or in rearrangements with DEB treatment. No quadriradial figures were seen in any of the BS fibroblasts. A single triradial configuration was the only chromatid interchange figure found. An increase in the frequency of telomeric association was noted.

Other Fibroblast Strains We previously reported [18] that fibroblasts from FA patients (C12, C17) had spontaneous chromosome breakage levels of 0.20-0.36 breaks per cell. Treatment with DEB resulted in a 3- to 5-fold increase in chromosome breakage in these cells. DEB was not found to have a clastogenic effect on XP cells (CRL 1158} or trisomy 18 cells (755), these being cells with low levels of spontaneous breakage. DISCUSSION What emerges from these studies is a remarkably nonuniform response within a small group of diseases, all of which have a genetic basis, increased cancer susceptibility, and a presumed origin in defective DNA repair or synthesis. Clear differences were found in the patterns of spontaneous and induced chromosome breakage in the different syndromes. The characteristic chromosomal abnormalities found in untreated FA and AT fibroblasts were achromatic lesions (chromatid and isochromatid gaps and breaksl, whereas in untreated BS cells, stable chromosome aberrations (rearrangements and deletions) were more frequent. The XP fibroblasts showed no spontaneous chromosome breakage. Exposure to a nontoxic (0.01 Izg/ml) concentration of DEB caused chromosome breakage and rearrangement in fibroblasts from both FA and AT patients, with FA cells having a greater susceptibility to the clastogenic agent. Similar increments in chromosome breakage were observed in AT strains chosen for their relatively high and low rates of spontaneous breakage. The same concentration of DEB had no clastogenic effect on fibroblasts derived from normal, BS, XP, or trisomic individuals. Using the same test system, we have reported that fibreblasts from FA heterozygotes, with essentially no spontaneous breakage, show increases in breakage proportional to those of fibroblasts from FA homozygotes [19]. Thus, the susceptibility to induced breakage by an exogenous agent is apparently

26

A.D. Auerbach and S. R. Wolman not related to the incidence of spontaneous breakage in the cell strain being tested. Our finding that AT cells have an increased sensitivity to chromosome damage by an alkylating agent contrasts with the normal response of AT lymphocytes to the induction of SCEs by other alkylating agents [20]. There is also a lack of correlation between the clastogenic effect and the induction of SCEs by alkylating agents in other chromosome instability syndromes. After stress with mitomycin C (MMC), FA lymphocytes have increased sensitivity to the induction of chromosome breakage, but show less than the normal increment of SCEs [21]. By contrast, the induction of SCEs by a variety of chemical agents is greater in XP fibroblasts than in normal cells [22], while the amount of chromosome breakage induced by some of these chemicals is normal. BS cells, which are the only cells from these syndromes to show increased base-line levels of SCEs [17], have increased sensitivity to SCE formation after exposure to the alkylating agent ethyl methanesulfonate [23], although DEB had no clastogenic effect in the present study. Thus the effect of clastogenic chemicals varies with the type of cell strain studied. The nature of cellular events resulting in chromosome breakage or SCEs awaits further elucidation. AT cells are highly sensitive to ionizing radiation, showing increased cell killing of both homozygous [24] and heterozygous [25] cells. Radiosensitivity is also observed at the chromosomal level, with the induction of an unusually high frequency of chromatid-type damage after G Oirradiation [5]. Although the defect in AT has not been well characterized as yet, defective repair of base damage resulting from high doses of anoxic y-irradiation has been shown [26,27]. AT cells are also sensitive to cell killing by certain alkylating agents [28], although there is heterogeneity in their response, just as there is when AT cells are assayed for repair of damage from yirradiation [27]. Treatment with alkylating agents, like ionizing irradiation, induces "short patch" repair [29], and our present finding in AT cells of increased chromosome damage from exposure to an alkylating agent is further evidence for defective repair in these cells. Although these syndromes are often lumped together as "chromosome instability syndromes" or "DNA repair deficient syndromes," it is important to recognize that the etiology of their increased chromosome fragility and susceptibility to clastogenic agents differs. Although the use of these cells has been suggested repeatedly for human mutagen screening [22, 27] cells from the different syndromes will have enhanced sensitivity to different agents for intrinsically different reasons. More important, however, is the interpretation of tests using "susceptible" cell strains. One must be cautious in extrapolating from data obtained from especially sensitive or susceptible cell strains; if used in a test system for human mutagens and carcinogens, such cells may respond in ways which are not representative of the more general population. If, as in the present study, the normal cell is resistant to chromosomal damage, even at semilethal doses of a carcinogen, then control of exposure to such an environmental agent should be directed towards identification and protection of the susCeptible subgroups within the human population. Note Added in Proof. AT cells have recently been shown to have unusual sensitivity to another radiomimetic chemical, bleomycin, as demonstrated by cytogenetic and cell survival methods [30]. The authors are grateful to Drs. M. Swift, J. German, and R. Cox for cell lines, Dr. B. Goldschmidt for providing them with DEB, and to Dr. J. Mitra for helpful discussions.

C a r c i n o g e n - i n d u c e d C h r o m o s o m e Breakage

27

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