Teratogenesis, Carcinogenesis, and Mutagenesis 18:279–291 (1998)

© 1998 Wiley-Liss, Inc.

Common Fragile Site Expression andGenetic Predisposition to Breast Cancer

Gül sah Çeçener, 1 Ünal Egeli, 2* I.smet Ta sdelen, 3 Berrin Tunca, 2

Hakan Duman, 3 and Ayhan Kizil 3

1Department of Molecular Biology, Uludag4 University College of Science,Bursa, Turkey

2Department of Medical Biology, Uludag4 University Medical College,Bursa, Turkey

3Department of General Surgery, Uludag4 University Medical College,Bursa, Turkey

The expression of common fragile sites induced by aphidicolin and caffeine wasevaluated on prometaphase obtained from the peripheral blood lymphocytes of 35women with breast cancer, their 35 clinically healthy female family members, and20 sex- and age-matched normal controls. As a result of the cytogenetic and statisti-cal evaluation, the number of damaged cells, chromosomal aberrations, and expres-sion frequencies of fragile sites detected in patients with breast cancer and theirfirst-degree relatives were found to be significantly higher than those in the controlgroup. Our findings indicate an increased genetic instability in women with breastcarcinomas and their relatives. Therefore, fragile sites may be used as a reliable markerfor defining genetic susceptibility to cancer in general. Teratogenesis Carcinog. Mu-tagen. 18:279–291, 1998. © 1998 Wiley-Liss, Inc.

Key words: breast cancer; fragile sites; genetic susceptibility; chromosomal abnormalities;aphidicolin; caffeine

INTRODUCTION

Genetic alterations play an important role in the development of sporadic andhereditary forms of breast cancer. Approximately 5–10% of all breast cancer is thoughtto be due to an inherited predisposition [1,2].

Common fragile sites are specific points on human chromosomes where gaps,breaks, or rearrangements are observed under specific culture conditions [3–8]. Theprecise relationship between fragile sites and cancer is not well established, eventhough it has been found that 67% of in vitro mutagen-induced fragile sites are coin-

*Correspondence to: Dr. Ünal Egeli, Department of Medical Biology, Uludag4 University Medical Col-lege, Bursa, Turkey.

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cidental with cancer-specific chromosome breakpoints and 72% are coincidental withoncogene loci. Therefore, fragile sites have been proposed to be involved in carcino-genesis [9–13].

The relationship between the presence of a high frequency in chromosomal ab-errations and predisposition to cancer has been well established in the “chromosomalinstability syndromes” [14] and this genomic instability is also present in a low fre-quency in peripheral blood lymphocytes in individuals affected by several types ofcancer [15,16].

Several investigations have revealed that there were great individual differencesin expression of common fragile sites and that the phenomenon was more frequentin normal cells of patients with certain types of malignancies [9,15,17,18] and infirst-degree relatives [19–21].

In the present study, fragile site induction in lymphocytes from patients withbreast cancer and their relatives was investigated and compared to a control group todetermine whether such patients exhibit an increased sensitivity to fragile site induc-tion by aphidicolin and caffeine, and whether, therefore, aphidicolin- and caffeine-induced fragile site expression in lymphocytes could be used as an assay to detectgenetic susceptibility to breast cancer.

MATERIALS AND METHODS

This investigation has been carried out on 35 patients with breast cancer, 35 oftheir first-degree female relatives, and 20 normal healthy women at the Genetic Labo-ratory of the Faculty of Science, Uluda� University. The diagnosis of the patients hasbeen made at the Department of Surgery of the Faculty of Medicine, Uluda� Univer-sity. A sample pedigree of one of our patient’s family is shown in Figure 1. Normalcontrols have been selected from persons without familial cancer history. In selectingthe subjects, we ascertained that they had not received X-irradiation, had not sufferedfrom a viral infection, and had not taken medications or drugs within the last 3 months.

The peripheral blood samples taken from patients and normal controls werecultured in RPMI 1640 medium (pH 7.2) containing 15% fetal calf serum, 6 mg/mlphytohemagglutinin L (PHA-L), 0.5 mg/ml L-glutamine, and antibiotics (100 IU/mlpenicillin, 100 mg/ml streptomycin) for 72 h at 37°C. Aphidicolin (0.2 mM) and caf-feine (2.2 mM) were added to the culture medium 5 h before the harvest for fragilesite expression. Ethidium bromide and colchicine were added to the medium 2 and 1

Fig. 1. Pedigree of the eleventh breast cancer patient’s family.

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h before the harvest, respectively. Chromosome preparations were made using a rou-tine method. The slides were stained with a Giemsa solution. The structural chromo-somal abnormalities in 30–50 metaphases belonging to each subject were counted bymeans of a light microscope (Zeiss axioplan, Thornwood, NY). After the metaphaseswith breaks were located on the slides, these slides were destained with methanol(Merck, Darmstadt, Germany). High-resolution banding (HRB) was performed todetermine the exact locations of gaps and breaks. A site was considered fragile if itappeared one or more times in 50 cells analyzed for each subject and in at least threeof all the subjects tested. A nonparametric statistical analysis (Mann-Whitney test)was used for comparison of the frequency of chromosomal aberrations and fragilesites in patients with breast cancer, their relatives, and controls.

RESULTS

The cytogenetic and statistical evaluation of the results related to patients withbreast cancer, their relatives, and the control group are presented in Tables I–IV.When the rates of chromosomal aberrations of patients and their relatives were com-pared with the control group they were found to be statistically significant. However,they were insignificant when the patients with cancer were compared to their rela-tives (Table III, Fig. 4). The separation of fragile sites in patients, their relatives, andcontrols is presented in Table IV. Total fragile site rates were determined to be 23.9%for the patients, 25.5% for the relatives, and 3.1% for the control group. When therates of cells with damage, chromosomal aberrations, and total fragile sites of pa-tients and their relatives were compared with those of the controls, they were foundto be statistically significant (Tables III, IV; P < 0.001). However, they were insig-nificant when the patients were compared to relatives (Tables III, IV; P > 0.05).

In the present study, 1p21, 1p32, 1p36, 1q21, 1q25, 1q42, 2p23-24, 2q21, 2q31-33, 2q37, 3p14, 4p15, 5q13, 5q31-33, 7p13-14, 7q32, 11p14-15, 13q12-13, 13q21,14q23-24, 16q21-23, 17q21, 18q21, 22q12 fragile sites were observed. When therates of 2q21, 2q31-33, 3p14, 7p13-14, 7q32, 13q21, 16q21-23, 17q21, 18q21, 22q12,and total fragile sites of patients and their relatives were compared with the controlgroup and were found to be statistically significant (P < 0.05). However, they werenot significant when the patients were compared to their relatives (except 1q25) (TableIV, Fig. 3; P < 0.05). Chromosomal localizations and condensations of fragile sitesare shown in Figure 2. Therefore, the sites of 1p36, 2q31-33, 2q37, 13q12-13, 13q21,and 17q21 were accepted as especially specific regions for breast carcinoma. Theseresults were significant since these are the sites of tumor suppressor genes BRCA1(17q21) and BRCA2 (13q12-13), putative tumor suppressor gene p73 (1p36), andmismatch repair gene hPMS1 (2q31-33).

DISCUSSION

Our results indicate that there is a relative increase in chromosomal instabilityin female patients with breast cancer and their first-degree female relatives (TableIV). The results also suggest that common fragile sites may be preferential points ofbreakage and that expression of these sites might be a primary contributor to chro-mosomal damage of the somatic genome. Recently, some investigators have pointedout that there are great personal differences in the expression of fragile sites and that

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TABLE I. Data on Paitents With Breast Cancer, Their Relatives, and Control Group*

Patients Relatives Controls

Subject Age Origin Localization Familial Subject Age Familial Subject Age FamilialNo. (years) Ductal Lobular In situ Invasive Infiltrative of tumor Stage antecedents No. (years) antecedents No. (years) antecedents

1 35 + – – – + R 3A + 1 40 M, A, U 1 53 –2 51 + – – – + L 3A – 2 25 M 2 73 –3 63 – + – – + R 1 – 3 47 M 3 44 –4 54 – _ – – + L 1 – 4 41 M 4 47 –5 72 + – – – + R 2A – 5 49 S 5 42 –6 37 + – – – + L 3A + 6 53 S 6 44 –7 53 + – – – + L 3A – 7 36 M 7 46 –8 40 + – – – + L 4 + 8 46 A, C, C 8 39 –9 60 + – – – + L 3A + 9 41 M, A 9 35 –

10 41 + – – – + L 2A + 10 40 M, U, B 10 48 –11 64 + – – – + R 2B + 11 31 M 11 47 –12 64 + – – – + R 3A – 12 21 M 12 33 –13 33 + – – – + R 1 + 13 38 A 13 40 –14 61 + – – – + R 2C – 14 51 S 14 24 –15 41 + – – – + R 2A – 15 14 M 15 25 –16 44 + – – – + R 2B – 16 49 S 16 26 –17 49 + – – – + L 3A – 17 32 M 17 39 –18 39 + – – – + L 1 – 18 19 M 18 44 –19 45 + + – + – R 1 + 19 39 S 19 34 –20 47 + + – + – R 1 – 20 48 S 20 34 –21 37 + – – – + R 2B – 21 30 S22 55 + – – – + R 0 – 22 56 S23 45 + – + + – L 3A – 23 35 A24 55 + – – – + R 0 – 24 46 M25 45 – – + – – R 1 – 25 42 M, C26 40 + – – – + R 1 – 26 48 S, S, C27 62 + – – – + L 1 + 27 29 M28 72 + – – + + l 2A – 28 51 M, GM29 49 + – – + – L 2B – 29 38 M30 47 – – + – – L 0 – 30 45 D31 41 + – – – – L 2A – 31 24 A, GF, U32 58 – + – + – L 1 + 32 24 M33 70 – + – – + L 2A – 33 48 M, A34 34 + – – – + L 1 + 34 42 M, U35 61 + – – – + L 2A + 35 48 A, A

*R, right; L, left; M, mother’s; S, sister’s; C, cousin’s; GF, grandfather’s; U, uncle’s; A, aunt’s; GM, grandmother’s; D, daughter’s; B, brother’s.

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TABLE II. Chromosomal Aberration Rates (Gaps and Breaks/Cell) of Patients With Breast Cancer, Their Relatives, and Control Group

Patients Relatives Controls

Subject Examined Damaged Subject Examined Damaged Subject Examined DamagedNo. metaphases cells Aberrations No. metaphases cells Aberrations No. metaphases cells Aberrations

1 50 0.66 1.18 1 50 0.18 0.36 1 30 0.07 0.072 50 0.56 0.94 2 30 0.53 1.20 2 30 0.07 0.073 40 0.35 0.63 3 40 0.20 0.23 3 50 0.08 0.104 50 0.16 0.16 4 50 0.18 0.30 4 30 0.17 0.235 50 0.26 0.54 5 50 0.42 1.24 5 30 0.06 0.066 50 0.06 0.08 6 50 0.44 0.78 6 50 0.14 0.147 50 0.22 0.20 7 30 0.10 0.10 7 30 0.03 0.038 40 0.08 0.08 8 30 0.33 0.77 8 30 0.03 0.039 50 0.34 0.54 9 50 0.34 0.92 9 30 0.13 0.13

10 50 0.44 0.62 10 40 0.35 0.80 10 30 0.13 0.1311 50 0.36 0.50 11 30 0.06 0.06 11 30 0.00 0.0012 30 0.54 1.00 12 30 0.00 0.00 12 30 0.00 0.0013 50 0.40 0.68 13 30 0.07 0.07 13 30 0.00 0.0014 30 0.50 1.05 14 30 0.23 0.26 14 50 0.00 0.0015 30 0.47 1.47 15 30 0.17 0.17 15 50 0.02 0.0016 30 0.47 1.20 16 30 0.06 0.06 16 50 0.04 0.0217 50 0.18 0.38 17 50 0.16 0.22 17 50 0.02 0.0218 40 0.05 0.05 18 40 0.05 0.05 18 50 0.00 0.0019 30 0.36 0.36 19 30 0.03 0.03 19 30 0.20 0.2020 40 0.20 0.30 20 40 0.05 0.05 20 50 0.00 0.0021 30 0.26 0.43 21 30 0.07 0.0722 30 0.13 0.17 22 30 0.05 0.0523 30 0.07 0.07 23 40 0.35 0.4824 30 0.20 0.16 24 50 0.32 0.5625 30 0.37 0.33 25 50 0.44 1.1826 50 0.20 0.32 26 40 0.45 0.8327 30 0.00 0.00 27 50 0.48 0.9828 50 0.48 0.68 28 50 0.36 0.4429 40 0.28 0.33 29 50 0.08 0.0830 50 0.42 0.92 30 40 0.43 0.5531 50 0.40 0.64 31 40 0.28 0.9032 40 0.33 0.33 32 40 0.13 0.1833 30 0.13 0.20 33 50 0.30 0.4834 50 0.18 0.22 34 40 0.30 0.7035 40 0.28 0.33 35 50 0.14 0.14

Mean ± SD 0.297 ± 0.164 0.488 ± 0.374 0.232 ± 0.156 0.437 ± 0.388 0.06 ± 0.06 0.06 ± 0.07

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these sites are more frequent in normal cells of patients with some types of neo-plasms [9,17–19,22–25] and sometimes in their first-degree relatives [19–21,24,25].Therefore, they consider that the expression of common fragile sites could be anindicator of chromosomal instability. However, some researchers here refused thisidea, because common fragile sites were found in all human genomes [22,26–28],and although common fragile sites are seen in all humans, their expression frequencyshows important differences between individuals. Essentially, this situation is char-acteristic in fragile site studies.

TABLE III. Comparison of Chromosomal Abnormalities Between Patients With Breast Cancer,The Relatives, and Control Group

P

Compared criteria Damaged cells Chromosomal aberrations

Patient-control <0.001 <0.001Relative-control <0.001 <0.001Patient-relative >0.05 >0.05

TABLE IV. Comparison of Fragile Sites Between Patients With Breast Cancer, Their Relatives,and Control Group

Fragile Mean ± SD

Sites Patients Relatives Controls

1p21 0.1494 ± 0.256* 0.01006 ± 0.01953 0.00165 ± 0.007381p32 0.00757 ± 0.01308 0.00451 ± 0.00939 0.00330 ± 0.010161p36 0.00946 ± 0.01336* 0.00446 ± 0.01019 0.00165 ± 0.007381q21 0.007 ± 0.01887 0.01063 ± 0.01918* 0.00165 ± 0.007381q25 0.00943 ± 0.01552* 0.00380 ± 0.01211 0.0000 ± 0.00001q42 0.00437 ± 0.00911 0.00494 ± 0.01136 0.00265 ± 0.008422p23-24 0.0717 ± 0.1394 0.00543 ± 0.01482 0.00495 ± 0.012092q21 0.00503 ± 0.01049* 0.00760 ± 0.01441* 0.0000 ± 0.00002q31-33 0.01943 ± 0.02919**** 0.01637 ± 0.2131**** 0.0000 ± 0.00002q37 0.00900 ± 0.01612 0.00734 ± 0.02362 0.00330 ± 0.010163p14 0.3120 ± 0.03959*** 0.03817 ± 0.05931* 0.00595 ± 0.012484p15 0.00417 ± 0.01080 0.00560 ± 0.01075* 0.0000 ± 0.00005q13 0.00209 ± 0.00715 0.00171 ± 0.00568 0.0000 ± 0.00005q31-33 0.00760 ± 0.1389 0.00740 ± 0.01510 0.00265 ± 0.008427p13-14 0.00546 ± 0.01158* 0.00657 ± 0.01547* 0.0000 ± 0.00007q32 0.00666 ± 0.01229* 0.00966 ± 0.02288* 0.0000 ± 0.000011p14-15 0.00629 ± 0.01573 0.00829 ± 0.01992* 0.0000 ± 0.000013q12-13 0.01000 ± 0.01857* 0.01294 ± 0.02123* 0.00165 ± 0.0073813q21 0.01300 ± 0.02041*** 0.01460 ± 0.02068**** 0.0000 ± 0.000014q23-24 0.01197 ± 0.02208* 0.01357 ± 0.03463 0.00165 ± 0.0073816q21-23 0.01343 ± 0.03169* 0.01763 ± 0.02062**** 0.0000 ± 0.000017q21 0.01323 ± 0.02363*** 0.01729 ± 0.01954**** 0.0000 ± 0.000018q12 0.00389 ± 0.00985 0.00337 ± 0.00988 0.0000 ± 0.000018q21 0.00646 ± 0.01447* 0.01309 ± 0.02267*** 0.0000 ± 0.000022q12 0.01023 ± 0.02225* 0.01046 ± 0.01791** 0.0000 ± 0.0000

Total 0.239 ± 0.222**** 0.255 ± 0.258**** 0.031 ± 0.062

*P < 0.05.** P < 0.01.*** P < 0.005.**** P < 0.001.

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Fig. 2. Localization of all aberrations observed in patients with breast cancer (a) and relatives (b). The solid circles indicate gapor breakpoints. The asterisks indicate breakpoints of one subject.

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Figure 2A.

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Fig. 3. The number of patients (a) and relatives (b) that show fragile sites.

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Fig. 4. The frequency of fragile sites in patients (a) and relatives (b).

Fragile Sites and Heritable Breast Cancer 289

However, Mitchell et al. [28] did not obtain meaningful results in a study per-formed with patients with familial breast cancer because their subject numbers weretoo few. Statistical tests are very important in scientific fragile site studies becausesignificance or nonsignificance can be determined according to these tests. It is wellknown that the reliability of statistical tests increases by increasing the subject num-bers. Our study was performed on 90 subjects (35 patients with breast cancer, 35 oftheir first-degree female relatives, and 20 healthy controls). Moreover, in our study,strict criteria were used to define fragile sites as mentioned in the Materials andMethods. Therefore, we believe that the reliability of our findings was increased.

We consider that there is a relationship between cancer-specific breakpoints andcommon fragile sites such as fra(3)(p14), (1)(p36), (2)(q31-33), (5)(q31-33), (16)(q22-23), etc. Recently, molecular genetic studies have shown that these sites involve tu-mor suppressor and mismatch repair genes, as well as oncogenes. In our study, thefra(3)(p14) expression was observed most frequently (Table IV). Moreover, the ex-pression of this site was observed in cancers such as lung, head and neck, colorectal,and ovarian [19,21,24,25]. Therefore, this site was considered one of the primarysites for all human cancers. Restriction fragment length polymorphism (RFLP) analysisof several tumor tissues indicates that all of them may have a deletion at chromo-some bands 3p14 and 3p21 [29]. It is known that in these sites FHIT putative tumorsuppressor gene and hMLH1 mismatch repair genes are localized, respectively [30,31].

In our study, the expression of some fragile sites was statistically significant inpatients and relatives compared with the controls (Table IV; P < 0.05). These siteswere fra(2)(q21), fra(2)(q31-33), fra(3)(p14), fra(7)(p13-14), fra(7)(q32), fra(13)(q12-13), fra(13)(q21), fra(16)(q21-23), fra(17)(q21), fra(18)(q21), and fra(22)(q12). There-fore, these sites were accepted to be specific sites for breast cancers. We consideredthese results to be significant, because these sites involved oncogenes, tumor sup-pressor genes, and mismatch repair genes.

It is known that tumor suppressor genes are very important for cancer forma-tion. BRCA1 and BRCA2 genes are responsible for breast cancer formation [32,33].In these genes, somatic or germ line mutations are known to cause breast cancerformation or predisposition. In our study, the fragility has been determined in sites17q21 and 13q12-13. This clue was considered clear evidence because of the appar-ent relation with cancer genes of fragile sites.

Aphidicolin is an inhibitor of DNA polymerase a and d, and it is directly in-volved with the DNA synthesis and replication process [34,35]. Therefore, the pres-ence of aphidicolin is likely to inhibit the DNA repair mechanism, leading tomaintenance of a series of chromosomal alterations caused by chromosomal instabil-ity. In our study, chromosomal damages and fragile site frequencies were not in-creased in the healthy female controls although the same dose of aphidicolin wasadministered to patients, relatives, and healthy females. A possible explanation forthis situation may be that control subjects were genetically stable, so that DNA syn-thesis and repair inhibitors such as aphidicolin had no particular effect on their chro-mosomal DNA.

Several investigators pointed out that patients with lung and head and neck can-cer and their first-degree generations might have an imbalance in their DNA repaircapacity [19,21,31,36]. Therefore, Liu et al. [19] and Egeli et al. [21] suggested thatthese individuals were shown to have genetic predisposition to cancer associatedwith the increasing fragile site expression. Recently, some molecular genetic studies

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revealed that germ line mutations had to be in mismatch repair genes such as hPMS1,hPMS2, hMLH1, and hMLH2 of patients with familial colorectal cancer [31]. Wedetermined fragility in 2q31-33 band regions located at hPMS1 gene in patients withcolorectal cancer and their first-degree generations [24,25]. It is clear that these frag-ile sites and mutations in mismatch repair genes could create a genetic predisposi-tion to colorectal cancer. In the present study, the fragilities were determined in bothhPMS1 gene localized in the 2q31-33 band and hMLH1 gene localized in the 3p21band. Therefore, the fragilities in hPMS1 and hMLH1 mismatch repair gene regionsmay create a susceptibility to mutations so that mutation incidence in oncogenes andtumor suppressor genes may increase and cause early breast cancer with familialrelations.

Consequently, common fragile sites may be unstable regions of human genome.Therefore, we consider that common fragile site expression may be a suitable markershowing predisposition to breast cancer and may be used for early diagnosis of can-cer. However, we believe that this type of studies must be supported with moleculargenetic studies.

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