Telomeric associations and chromosome instability in ataxia telangiectasia T cells characterized by TCL1 expression

Cancer Genetics and Cytogenetics 125 (2001) 46–51

Telomeric associations and chromosome instability in ataxia telangiectasia T cells characterized by TCL1 expression Paola Petrinellia,*, Raffaella Ellia, Liana Marcuccia, Elisabetta Tabolaccia, Concetta Barbierib, Anna Antonellia a

Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università “La Sapienza”, Rome, Italy b Servizio di Allergologia e Immunologia Pediatrica, Università “La Sapienza”, Rome, Italy Received 9 May 2000; received in revised form 11 August 2000; accepted 28 August 2000

Abstract

T-cell tumors in ataxia telangiectasia (AT), such as T-PLL/T-CLL, are first preceded by the development of a large clone of T-lymphocytes, characterized by chromosomal rearrangements, which usually involve specific regions such as the 14q11 region. Malignancy develops years later, after additional chromosomal changes resulting from the genomic instability consequent to ATM disruption and to the activation of the TCL1 oncogene. Here we report the results of a cytogenetic followup of an AT patient (AT94-1), still without signs of hamatological abnormalities, bearing a T-lymphocyte clone characterized by the t(14;14)(q11;q32) rearrangement and having TCL1 expression. We demonstrated that in clonal cells TCL1 expression correlates with increasing genomic instability and in time this mainly induces chromosomal rearrangements and telomeric associations (tas). Chromosome 21 is not randomly involved; in particular, an i(21q) indicates that it is a subclone prone to additional genetic changes and could represent an early chromosomal rearrangement involved in tumorigenesis. With regard to the increase in tas, we observed that: (i) it is inversely correlated with the proliferative ability of AT94-1 lymphocytes in PHA-stimulated short-term cultures (cell aging in vitro); (ii) this increase is not due to changes either in cell radiosensitivity (measured as bleomycin (BML)-sensitivity) or due to an illegitimate recombination (measured as adriamycinsensitivity), which may not be sufficient for tumor development. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction Ataxia telangiectasia (AT) is a recessive inherited disorder caused by mutation of the ATM gene (mapped to chromosome 11q22.3), which codes a protein characterized by phosphatidil inositol 3-kinase domain and involved in DNA damage processing and cell cycle control [1–3]. It has been suggested that the frequent malignancies, especially lymphoid, observed in AT patients are a consequence of the genomic instability associated with the syndrome. The involvement of the ATM gene also has been shown in the rare sporadic disease T-cell prolymphocytic leukemia (T-PLL) [4] and in a far more frequent sporadic disease, B-cell lymphocytic leukemia (B-CLL) [5]. Therefore, ATM could act as a tumor suppressor gene, at least in the development of T-PLL [6]. However, ATM disruption is not the only necessary event in T-PLL tumorigenesis. Another equally important event is likely to be the activation in the * Corresponding author. Tel.: +39-06-4469843; fax: +39-06-4462891. E-mail address: [email protected] (P. Petrinelli).

T-lymphocytes of the TCL1 oncogene by juxtaposition with the T-cell receptor alpha/delta locus, as a result of chromosomal rearrangements such as: t(14;14)(q11;q32), t(7;14)(q35; q32) or inv(14)(q11q32) [7,8]. Cells bearing these rearrangements can show clonal expansion with time and transform into leukemic cells, both in AT patients and normal individuals [9]. We have previously reported the expansion (4% to 66%) of a cellular clone, bearing the tandem t(14;14)(q11;q32) rearrangement correlated to TCL1 expression, in the T-lymphocytes of an AT patient (AT94-1) [10,11]. At the time of the study, the patient showed no signs of hematological disease and continues to be in this status today. Here we report the results of the cytogenetic follow-up in these cells, which have served as a useful model to study the role of chromosome instability in neoplastic transformation. Since 1995, we have studied spontaneous chromosome instability separately in the two AT94-1 cell subpopulations (with and without the tandem translocation). During this study we observed an increase in the frequency of telomeric association (tas), almost exclusively in

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Table 1 Cytogenetic follow-up of AT94-1 lymphocytes Metaphases with t(14;14)

Date

No. cells analyzed

Metaphases without t(14;14)

Total

I (%)

II (%)

III (%)

Total

I (%)

II (%)

III

1995 1996 1997 1999 2000

35 57 36 72 60

28 46 25 54 53

21 (75) 34 (74) 20 (80) 32 (59) 29 (55)

5 (18) 11 (24) 2 (8) 11 (20) 15 (28)

2 (7) 1 (2) 3 (12) 11 (20) 9 (17)

7 11 6 16 7

7 (100) 10 (91) 6 (100) 13 (81) 6 (86)

0 1 (9) 0 3 (19) 1 (14)

0 0 0 0 0

I: metaphases without rearrangements; II: metaphases with rearrangements; III: metaphases with at least one telomeric association (tas).

clonal cells. The meaning of this kind of rearrangement in AT cells is being debated. In fact, although AT lymphocytes, have normal telomerase activity, they show accelerated shortening of telomeres and a high frequency of tas [12,13]. We attempted to establish whether the increase of tas in AT94-1 cells overexpressing the TCL1 oncogene may be a consequence of: (i) changes in growth rate, (ii) an increase in radiosensitivity and/ or in illegitimate recombination activity. Thus we investigated: (i) the proliferative ability of these lymphocytes in PHA-stimulated short-term cultures; (ii) their sensitivity to the radiomimetic drug bleomycin (BLM) and to the DNA topoisomerase II poison adriamycin (ADR). The former mainly induces chromosome breakage, whereas the latter, in addition to inducing chromosome breaks, specifically induces exchange figures that can be taken as a marker of illegitimate recombination. 2. Materials and methods The AT94-1 patient is now 20 years old; her main clinical features and T-cell immunophenotype data have been reported previously [10,11]. In the last 5 years, hematological parameters have been routinely monitored and have shown normal values. The patient has not had any particular infectious episodes. Precocious greying of hair was noted whereas cutaneous hyperpigmentation and skin atrophy are not present.

concentrations 10 and 30 ␮g/ml) was added to cultures 4 h before harvesting; alternatively ADR (Pharmacia and Upjohn), diluted in isotonic solution (final concentration 60 ng/ ml), was added to cultures 24 h before harvesting. Chromosome breakage was blindly evaluated by two independent observers. The classification of metaphases was based on five levels of damage: undamaged cells, one or two breaks/cell, more than two breaks/cell, partial or full metaphase pulverization, and one or more exchange figures. The chi-square test (RxC table) was used for statistical analysis of the chromosome damage distribution. 3. Results 3.1. Cytogenetic follow-up We performed the sequential cytogenetic analyses on the AT94-1 lymphocytes over a period of 6 years (1995–2000). The frequency of clonal cells bearing the tandem translocation t(14;14)(q11;q32) was stable in time with a value ranging from 70% to 88%. Table 1 shows the frequency of normal and abnormal metaphases in clonal and non-clonal

2.1. Lymphocyte cultures Peripheral lymphocytes are obtained from the AT94-1 patient, from 3 normal controls and 2 AT controls (AT95-1, AT95-2) and were cultured by standard methods for 72, 96, and 120 h. AT95-1 and AT95-2 were reported as AT22RM and AT28RM, respectively, by Gilad et al. [14]. 2.2. Chromosome instability The analysis of spontaneous chromosome rearrangements was performed on at least 30 G-banded metaphases from the 72-h cultures. The spontaneous rate of tas was evaluated analyzing at least 100 Giemsa-stained metaphases from the 72, 96, and 120 h cultures. In particular, the identification of the chromosomes involved was performed on at least 35 G-banded metaphases from the 72 h cultures. To analyze induced chromosome instability, bleoryein (BLM) (Nippon Kayaku), diluted in distilled water (final

Fig. 1. Metaphase G-banded from clonal AT94-1 lymphocytes showing telomeric associations. Small arrows indicate tas (21;22)(p1;q13) and tas (18;21)(p11;q22); big arrow indicates the chromosome resulting from t(14; 14)(q11;q32).

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P. Petrinelli et al. / Cancer Genetics and Cytogenetics 125 (2001) 46–51

Table 2 Follow-up of spontaneous chromosome rearrangements in AT94-1 clonal cells Date Subclones

Structural anomaly

1995 45,XX,t(14;14)(q11;q32),⫺22,⫹mar1[1] 44,i(21)(q10),⫺6[1]

a

del(1)(q32)[1] der(14)t(14;14)(q11;q32)add(14)(q32)[1] idic(12)(q24)[1] 1996 45,XX,t(14;14)(q11;q32),i(21)(q10)[2] del(16)(p10)[1] 45,XX,t(14;14)(q11;q32),i(21)(q10),⫹8,⫺18,t(2;17)(q31;p?)[1] del(5)(p10)[1] 46,XX,t(14;14)(q11;q32),⫹8[1] del(15)(q?)[1] 45,XX,t(14;14)(q11;q32),⫹8,⫺22[1] i(6)(q10),inv(20)(p11q11)[1] 45,XX,t(14;14)(q11;q32),⫺22,⫹mar1[1] t(2;5)(q?;p?)[1] 1997 45,XX,t(14;14)(q11;q32),i(21)(q10),inv(7)(p14q35)[1] t(1;10)(q44;q11)[1]

1999 45,XX,t(14;14)(q11;q32),i(21)(q10)[5] 45,XX,t(14;14)(q11;q32),i(21)(q10),der(14)t(14;14)(q11;q32) add(14)(p13),del(15)(q22)[1] 45,XX,t(14;14)(q11;q32),i(21)(q10),t(4;22)(q35;q11)[1]

dup(22)(q11qter)[1] ⫺21,⫹mar2[1] t(2;21)(p13;q11)[1] t(5;15)(q11;q26)[1]

2000 45,XX,t(14;14)(q11;q32),⫺22,⫹mar1[1] 45,XX,t(14;14)(q11;q32),i(21)(q10)[4] 45,XX,t(14;14)(q11;q32),i(21)(q10),i(9)(q10)[1] 45,XX,t(14;14)(q11;q32),i(21)(q10),tas(3;16)(q29;q24)[1]

del(12)(p?)[1] del(16)(q?)[1] del(X)(q21)[1] ⫺21,⫹mar3[1] t(5;9)(q33;q34)[1] t(2;13)(q33;p11)[1] t(2;18)(p10;p10)[1] inv(17)(p13q24)[1]

Telomeric associations tas(2;5)(q37;p15)[1] tas(2;19)(q37;p13)[1] tas(9;15)(p24;q26)[1]

tas(19,21)(p13;q22)[1] tas(X;12)(q28;24)[1] del(5)(q31),tas(X;22)(p22;p13)[1] tas(11;12)(q25;q24),⫺20[1] tas(21;22)(p1;q13)[2] tas(5;21)(p15;p1)[1] tas(16;21)(q24;p1)[1] tas(2;4)(q37;p16)[1] tas(9;19)(q34;q13)[1] tas(7;19)(q36;q13)[1] tas(21;21)(p1;q22)[1] tas(4;13)(q35;p1)[1] inv(7)(p14q35),tas(13;21)(p1;p1)[1] tas(5;15)(p15;p1)[1] tas(5;6)(p15;p25)[1] tas(5;22)(p15;p1)[1] tas(3;16)(q29;q24)[1] tas(4;21)(q35;p1)[1] tas(X;2)(q28;q37)[1] tas(2;15)(q37;p1),tas(21;21)(p1;p1)[1] tas(21;22)(p1;q13),tas(18;21)(p11;q22)[1] add(22)(q?),tas(X;8)(p22;q24)[1]

Previously reported as “45,XX,t(14;14)(q11;q32),⫺22,⫹C-like” [11].

a

cells. Data show that: (a) the frequency of cells with chromosome rearrangements (II) or tas (III) was always significantly (P⬍.01) higher in clonal cells than in non-clonal cells; (b) the frequency of mitoses carrying tas increased with the patient aging (from 7% when 15 years old to 17% when 20 years old); (c) the frequency of clonal cells bearing t(14;14)(q11;q32) as the only chromosomal rearrangement decreases in time. This is not surprising as it is well established that such stable aberrations do decrease with time [15]. Fig. 1 shows examples of tas. The qualitative analysis of chromosome rearrangements and tas observed in cells bearing t(14q14q) is shown in Table 2. The two subclones identified in 1996 [11], 45,⫺22,⫹mar1 (previously reported as 45,⫺22,⫹ C-like) and 45,i(21)(q10), are still present. In particular, the 45,⫺22,⫹mar1 did not show expansion, whereas the i(21)(q10) subclone triplicated its frequency (from 3.4% to 11%) and 35% of its cells showed additional chromosome rearrangements. Chromosome 21 is involved in different types of rearrangements, both clonal [such as the subclone i(21)(q10)] and sporadic (such as translocations and tas). Hence, involvement is thought to be non-random. The isodicentric chromosomes observed in clonal cells in 1995 and the subclone with trisomy 8 reported in 1996 were no longer present.

3.2. Tas and aging of cultured AT94-1 lymphocytes We analysed the proliferation rate of clonal cells together with tas frequency at different times after PHA-stimulation. The results obtained with lymphocytes of the patient taken in two successive years showed an inverse correlation between survival and incidence of tas (Fig. 2). 3.3. Tas and AT94-1 lymphocyte sensitivity to DNA damage We tested AT94-1 lymphocyte radiosensitivity by evaluating BLM-induced chromosome instability (Table 3); the protocol used allowed us to highlight the damage induced in the S/G2 phase of the cell cycle (mainly chromatid type damage). The distributions of chromosome breakage observed in several assays over a period of 7 years were not significantly (P⬎.05) different from each other demonstrating that the radiosensitivity of total AT94-1 lymphocytes has not significantly modified over time. Moreover, as we have previously reported, BLM-induced chromosome damage is not significantly different in clonal and non-clonal cell subpopulations (data not shown). In addition, the frequency of tas scored in the different years in metaphases from BML-treated cultures is not significantly (P⬎.05) different from the data on the untreated cultures reported in Table 2.

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Table 3 BLM-induced chromosome instability of AT94-1 lymphocytes BLM dose Date 10 ␮g/ml 30 ␮g/ml

b

1988 1994b 1999 1995 1999

Cells with breaks (%)a

Undamaged No. cells (%)

a

b

c

100 167 129 88 148

30 (30) 57 (34) 38 (29) 24 (27) 49 (33)

32 (32) 45 (27) 33 (26) 43 (49) 58 (39)

0 0 3 (2) 6 (7) 8 (5)

38 (38) 65 (39) 55 (43) 15 (17) 33 (22)

a a: metaphases with one or two breaks; b: metaphases with more than two breaks; c: metaphases pulverized. b Previously published [11].

4. Discussion T-cell tumors in AT described in the literature, such as T-PLL/T-CLL, are first preceded by the development of a large clone in T-lymphocytes characterized by rearrangements such as t(14;14)(q11;q32), t(X;14)(q28;q11), t(7; 14)(q35;q32), and inv(14)(q11q32). Malignancy develops some years later after additional genetic changes [15]. Therefore, part of our study focused attention on chromosome modifications additional to the primary chromosome rearrangement in lymphocytes of a patient carrying t(14; 14)(q11;q32) who has still not developed tumors. Trisomy 8 (⫹8) is frequently present in hematological disorders and, in particular, amplification of genes on 8p may play a role in the pathogenesis of myeloid disorders [16]. Although anomalies of chromosomes 8 and 6 (trisomy 8q, i(8), 8p⫹, 6q⫺) are also reported in AT and non-AT patients who have developed T-PLL [15], we observed only a transient subclone characterized by full trisomy 8. Only chromosome 21 seemed not randomly involved in structural rearrangements. One of these, i(21)(q10), resulted in a subclone of the t(14;14) cells. And i(21q) was also reported in an AT patient (AT5 BI) carrying inv(14)(q12q32), who then developed T-PLL [17] and in non-AT subjects suffering from different types of

Fig. 2. Proliferation rate and frequency of tas in clonal cells bearing t(14;14). Proliferation rate is the ratio of clonal cells number/total cell number (closed symbols: 䊉, 1997; 䊏, 2000). The frequency of tas is the ratio of tas number/clonal cell number (open symbols: 䊊, 1997; ⵧ, 2000).

We tested the proneness of AT94-1 lymphocytes to illegitimate recombination by comparing the chromosome instability induced by ADR in these cells with that induced in lymphocytes from three normal subjects and from two AT subjects (AT95-1 and AT95-2) who do not show clonal rearrangements (Table 4). Statistical analysis of induced chromosome damage distribution shows that: (a) total AT94-1 lymphocytes are significantly (P⬍.01) more sensitive than normal cells and not significantly (P⬎.05) different from AT controls; (b) the ADRhypersensitivity of AT94-1 lymphocytes does not change with patient aging; and (c) AT94-1 clonal lymphocytes are significantly (P⬍.01) more sensitive than AT94-1 non-clonal lymphocytes (mainly in terms of exchange figures, resulting from illegitimate recombination). Moreover, clonal lymphocyte frequency is significantly reduced (from 80% to 55–60%) after ADR-treatment.

Table 4 Adriamycin-induced chromosome instability in AT94-1 lymphocytes, normal and AT control lymphocytes Date

Cells

No. cells analyzed

1995

AT94-1 (total) ⫹14q;14q ⫺14q;14q AT94-1 (total) ⫹14q;14q ⫺14q;14q AT94-1 (total) ⫹14q;14q ⫺14q;14q AT95-1 AT95-2 Normalc

312 144 117 111 57 37 141 72 46 86 130 481

1998

1999

a

Undamaged (%) 40 (13) 14 (10) 26 (22) 11 (10) 3 (5) 8 (22) 17 (12) 4 (5) 13 (28) 13 (15) 18 (14) 192 (39ⴞ2.5)

Cells with breaks (%)a a

b

Cells with exch. (%)

Heavily damaged (%)b

85 (27) 27 (19) 51 (44) 26 (23) 11 (19) 15 (40) 26 (18) 12 (17) 14 (30) 17 (20) 17 (13) 150 (32ⴞ2)

45 (14) 34 (24) 18 (15) 16 (14) 10 (18) 6 (16) 28 (20) 13 (18) 15 (33) 15 (17) 25 (19) 18 (4ⴞ0.5)

89 (29) 67 (47) 22 (19) 41 (37) 33 (58) 8 (22) 47 (33) 43 (60) 4 (9) 36 (42) 63 (48) 121 (25ⴞ2)

53 (17)

17 (15)

23 (16)

5 (6) 7 (5)

a: metaphases with one or two breaks; b: metaphases with more than two breaks. “Heavily damaged” is used to describe metaphases in which most of the chromosomes are not recognizable; therefore, it was not possible to distinguish clonal from non-clonal AT94-1 lymphocytes, based on the presence of the derivative chromosome resulting from the t(14;14). c Mean ⫾ SE of pooled data from three normal donors. b

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leukemic and preleukemic disorders [18,19]. In the patient here reported, the i(21)(q10) subclone seemed prone to additional genetic changes involving other chromosomes (5 of 18 cells with i(21)(q10) showed genetic changes during the 6-year follow-up). Therefore, isochromosome 21q formation, leading to trisomy 21q, (i.e., to the gain of an additional copy of the 21q22 region in which several genes involved in cell growth and differentiation, such as AML1, ERG and ETS genes, are mapped [20]), may be an early chromosomal change involved in tumorigenesis in these cells. This may be supported by the observation that individuals with Down syndrome have a high risk of leukemia and that their lymphocytes show an increased rate of telomere loss that may be due to a higher turnover of cells with trisomy 21 [21]. A further point of interest to come out of our follow-up is the increase in tas observed only in AT94-1 clonal cells and not observed in non-clonal cells of the same patient or in the control AT lymphocytes. The frequency of tas increased with the patient’s age. This correlates well with our observation that clonal cells proliferate and age faster than nonclonal cells and show progressive increase in tas, as could be expected in cells aging in vitro [22]. Tas increase has not led to tumorigenesis in this patient, in agreement with literature data showing no role in tumorigenesis for telomeric loss and/or tas in AT preleukemic clones. In fact, tas associations have not been observed in leukemic cells from AT patients carrying large clones and developing T-PPL [13]. The excess in tas observed cannot be attributed to the specific kind of ATM mutation in the AT94-1 patient. The mutation of the ATM gene in the AT94-1 patient, referred to as AT22RM [14], determines protein truncation with the loss of the ATM kinase domain and conservation of the leucine zipper (LZ) domain. As reported by Smilenof et al. [23], this type of mutation is thought to be correlated with increased telomeric association. Since the clonal and non clonal cells of AT94-1 share the same ATM mutation, our observation that only clonal cells show a high frequency of tas is in disagreement with Smilenof’s hypothesis [23], even taking into account the quite different experimental conditions. Lastly, the increase in tas observed in AT94-1 clonal cells is not related to other pleiotropic effects of ATM mutation, such as cellular radiosensitivity and recombination repair ability. Indeed, the distribution of chromosome damage induced in clonal cells by the radiomimetic drug BLM and by the DNA topoisomeraseII inhibitors VP16 (data previously reported) and ADR (present report) have not significantly changed since 1994–1995. It is worth noting the high susceptibility of AT cells, especially clonal cells, to DNA damage induced by ADR. This susceptibility mainly takes the form of exchange figures, which usually result from chromosome breakage and illegitimate recombination events. This is in agreement with the kind of molecular damage specifically induced by BLM and ADR [24]. In conclusion, the increase in tas we observed in this cy-

togenetic follow-up is not sufficient to account for leukemogenesis and cannot be taken as a marker of susceptibility to induced damage; rather, it can be regarded as a marker of cell aging in these clonal AT cells showing TCL1 expression.

Acknowledgments We would like to thank M. Proietti for his valuable technical participation in this work. This study was supported by a grant from MURST.

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