Leukemia Research Vol. 16, No. 6/7, pp. 681 691, 1992. 0145 2126/92 $5.00 + .00 Printed in Great Britain. Pergamon Press Ltd

M O L E C U L A R A N A L Y S I S O F A N A T A X I A T E L A N G I E C T A S I A

T - C E L L C L O N E W I T H A C H R O M O S O M A L T R A N S L O C A T I O N

t ( 1 4 ; 1 8 ) - - E V I D E N C E F O R A B R E A K P O I N T IN T H E T - C E L L

R E C E P T O R d i -CHAIN G E N E *

MICHAEL UPPENKAMP, IRINA GANA DRESEN, RE1NHARD BECHER,t MARK RAFFELD:~ and PETER MEUSERS

Department of Internal Medicine, Division of Hematology, University of Essen, Hufelandstr. 55, 4300 Essen, F.R.G., t West-German Tumor Center, University of Essen, F.R.G. and ~ National Cancer Institute, Department of Pathology, Section of Hematopathology, NIH, Bethesda, MD,

U.S.A.

(Received 3 June 1991. Revision accepted 19 October 1991)

Abstract--We established a clonal T-cell line with a reciprocal chromosomal translocation t(14;18)(q11;q23) from a patient with ataxia telangiectasia (AT) and T-cell chronic lymphocytic leukemia (T-CLL). The tumor cells and the derived T-cell line were compared with respect to phenotype, karyotype, and rearrangement pattern. Restriction fragment analyses of the T-cell receptor (TCR)-6 gene, which is located within the TCR-o: gene on chromosome 14qll, indicated that the breakpoint is located within the TCR-6 locus, splitting the TCR-6 gene between the variable and joining segments. This specific chromosomal translocation was only detected in the derived T-cell line and may be involved in the genesis of T-cell malignancies in AT.

Key words: Ataxia telangiectasia, T-cell lymphocytic lymphoma, reciprocal chromosomal trans- location, T-cell receptor gene rearrangements, T-cell receptor di-chain gene.

INTRODUCTION

.ATAXIA telangiectasia (AT) is a rare autosomal recessive disorder characterized by progressive cere- bellar ataxia, oculocutaneous telangiectases, and a primary immunodeficiency syndrome. Patients with AT show frequent chromosomal breakage and have an elevated risk of developing malignancies, pre- dominantly malignant lymphomas and leukemias [1]. Some of the malignant lymphomas and leukemias are associated with specific chromosomal aberrations involving sites, that correspond to T-cell receptor (TCR)- and immunoglobulin chain genes [2, 3].

Chromosomal translocations in AT patients with T-cell neoplasias often involve the T-cell receptor (TCR)-tr chain gene located on chromosome 14q11, which is a common chromosomal breakpoint in T- cell leukemia, particularly chronic lymphocytic leu- kemia, adult T-cell leukemia, and prolymphocytic

* This paper is dedicated to Prof. Dr G. Brittinger on the occasion of his 60th birthday.

Abbreviations: A T, ataxia telangiectasia; T-CLL, T-cell chronic lymphocytic leukemia; IL-2, interleukin 2; PHA, phytohemagglutinin; TCR, T-cell receptor; V, variable; D, diversity; J, joining; C, constant; mAb, monoclonal antibody; MBP, myelin basic protein.

681

leukemia [4-11]. This locus is of specific interest since the TCR-6 gene, which codes for the TCR-6 chain of the second 1I-6 T-cell receptor, is nested between Vo:- and Jet-region genes [12, 13]. The unique local- ization within the TCR-cr gene coupled with the early utilization of the T-y gene in T-cell ontogeny long before the TCR-c~ gene is rearranged makes it a preferred site of chromosomal breakage in T-cell lymphomas and leukemias [14, 15]. Recent sequence analyses mapped breakpoints involving 14qll to the TCR-6 gene locus, providing further evidence that the TCR-6 gene is involved in various T-cell-specific chromosomal translocations [15, 16].

Here we report of a reciprocal translocation t(14;18)(qll;q23) in a clonal T-cell line derived from a patient with AT and chronic lymphocytic leukemia of T-cell origin (T-CLL). Restriction fragment analy- sis using a J61-probe revealed an atypical rearrange- ment pattern, which could not be ascribed to either V6 gene usage or allelically excluded rearrangements seen in other cases [17, 18]. The data suggest that the rearrangement is due to a chromosomal translocation splitting the TCR-6 locus between the V- and J- regions with translocation of the J-C region to chro- mosome 18. This reciprocal translocation occurred in a minor T-cell clone that has not been derived

682 M. UPPENKAMP et al.

from the major T-cell clone in the patient. The trans- location may represent a premalignant lesion.

M A T E R I A L S AND M E T H O D S

Establishment and maintenance of cell line Peripheral blood mononuclear cells of a patient with AT

and T-CLL were separated by Lymphoprep (Nycomed Pharma, Oslo, Norway) density centrifugation and adjusted to 0.5 × 106/ml in RPMI 1640 (GIBCO-BRL, Grand Island, NY, U.S.A.) supplemented with 15% fetal calf serum (FCS), 2% human AB serum, and penicillin (100IU/ml)-streptomycin (100~tg/ml). Cells were cul- tured at a cell count of 103/ml with phytohemagglutinin (PHA; 10~tg/ml) (Sigma Chemical, St. Louis, MO, U.S.A.) in suspension for up to 3 days. Subsequently the cells were washed with RPMI 1640 and stimulated with recombinant interleukin 2 (IL-2; 1000 U/ml) (Cetus Corp., Emoryville, CA, U.S.A.) until cells proliferated. Recom- binant IL-2 (1000 U/ml) was critical for further cell growth. Cells were expanded and analysed in subsequent studies.

Karyotype analysis Lymphocytes were stimulated with PHA (Sigma),

harvested after 72 h and treated with colcemide (0.02 ~tg/ ml) for the last 2 h. After hypotonic treatment with KC1 (0.075 mol/l) cells were fixed in methylalcohol acetic acid (3 : 1). G-banding was performed according to conventional methods after short-term treatment with trypsin. Karyo- typic interpretation was based on the nomenclature pro- posed by the Paris Conference 1971.

Cell staining and monoclonal antibodies Viable cells were stained with monoclonal antibodies

(mAb) for surface expression of antigen markers and ana- lysed by indirect immunofluorescence using a FACS (FACS II, Becton Dickinson, Mountain View, CA, U.S.A.) [19]. Murine anti-human mAb utilized included OKT6 (CD1), OKT9 (CD71) (Ortho Diagnostic Systems, Raritan, NJ, U.S.A.), Leul (CD5), Leu4 (CD3), Leu2A (CD8), Leu3A (CD4), anti-interleukin 2-receptor (CD25), HLA-DR, Leul2 (CD19), Leul6 (CD20) monoclonal IgG, A, M, D, K, ), (Becton Dickinson), J5 (CD10) (Coulter Immunology, Hialeah, FL, U.S.A.), Lyt3 (CD2) (New England Nuclear-Dupont, Boston, MA, U.S.A.), and 3A1 (CD7) (American Type Culture Collection (ATCC), Rock-

ville, MD, U.SoA.). Mouse ascites (1:1000) (Bethesda Research Laboratories, Gaithersburg, MD, U.S.A.) ser- ved as a negative control.

Analysis of DNA High molecular weight DNA was isolated from 2 × 108

cells by ultracentrifugation using a guanidinium-iso- thiocyanate cesium-chloride gradient [20]. Restriction digestion with endonucleases EcoR1, BamH1, Hindlll, Kpnl, Sstl, and Xbal was performed as recommended by the manufacturer (BRL). DNA (15 ~tg) was size-fractioned by electrophoresis in 0.8% agarose gels, transferred to nylon membranes (Gene Screen Plus, NEN-Dupont) and hybridized with random primed 32p-labeled probes at a specific activity of 300-500 cpm/pg according to previously published protocols [21]. Nylon membranes were washed as recommended by the supplier and autoradiographed by standard techniques.

Probes The following probes were used: the C region of the

TCR-fl chain gene (Cfl; Aval-Pstl fragment obtained from cDNA YT35, provided by T. W. Mak, University of Toronto, Canada) [22]; a genomic fragment including the J region of the human T-7 gene (JT; 700 bp Hindlll-EcoRl fragment of M13H60, provided by T. Rabbitts, Cam- bridge University, U.K. [23, 24]. Rearrangements of the TCR-o:/b gene were detected using a C region probe of the human T-6 gene (3'C6:700 bp EcoRI-EcoRl fragment obtained from cDNA Pr81, provided by E. Loh, Stanford University, CA, U.S.A.) [25]; a genomic fragment of the J region (Jb; 900bp Sacl-Xbal fragment of GTH311) (provided by D. Cohen, NIH, MD, U.S.A.) [26]. Fur- thermore a Vbl-region probe (V61; 300bp EcoR1-Scal fragment of cDNA 0-240/47, provided by M. Krangel, Harvard Medical School, Boston, MA, U.S.A.) [27], and a 2 kb genomic fragment 5DHR7 that recognizes an EcoRl- Hindlll fragment located between D62 and D63 (M. Raffeld, personal communication) (Fig. 1). The myelin basic protein gene (MBP) that maps to chromosome 18q22- 23 was analyzed with genomic probes: a 1.2 Kb EcoR1- EcoR1 fragment of cDNA clone HBP-1 and a 940bp EcoR1-EcoR1 fragment of cDNA clone HBP-2 (ATCC) [28, 29].

RESULTS

Cell lines

Four cell lines were derived from the fresh tumor

pV`51

R R

-i ll D 7`51 7,52

/ h > 23 kb

SDH..._R7 P J J 1

B H BR R HH HX RX H B B

/ i D`511~2 D`53 J`51 J 82 J 83 0`5 V`53 Jot

-'7'/

FIG. 1. Organization and partial restriction map of the TCR-6 gene locus. This map shows location of probes (p) used in this study and relevant BamHl (B), EcoR1 (R), Hindlll (H), and Xbal (X) restriction enzyme sites. The location of TCR-6 region genes (V, D, and C) as well as

one Jo~ segment are shown.

I--4 1 kb

//-

Molecular analysis of an ataxia telangiectasia T-cell clone 683

cell population, designated 1795, upon IL-2- and PHA-stimulation. They exhibited an identical clonal rearrangement pattern by restriction fragment analy- sis, indicating that they were derived from the same T-cell clone. No additional rearranged fragments were detected in the four cell lines. Therefore karyo- typic analysis and subsequent studies were performed on cell line 1795/4A, which was the first celt line established.

lmmunophenotype Surface phenotype of the fresh tumor cell popu-

lation 1795 and the established cell line 1795/4A were determined by flow cytometry (Table 1). Both were of T-cell origin with a CD3-positive phenotype. No surface membrane immunoglobulins or other B-cell antigens (CD19) were expressed on the cells, except for a small percentage (2%) of CD20-positive cells in the fresh tumor material 1795. All cells were characterized by the early T-cell antigen CD7. Tumor cells 1795 coexpressed helper-inducer (CD4) and suppressor-cytotoxic (CD8) antigens on the surface membrane. Cell line 1795/4A displayed a pure helper-inducer T-cell phenotype. The coexpression of CD4 and CD8 of 1795 resembled a phenotype of common thymocytes, whereas cell line 1795/4A exhibited a mature T-cell phenotype (CD3 +/CD8 +) [30]. This T-cell phenotype usually correlates with cell surface TCR-o:/fl-protein expression in humans [31]. Cell line 1795/4A and also the tumor cells 1795 were CD5 antigen-positive, expressed HLA-DR and CD25, and lacked CD1 and CD10 antigens. A high percentage of 1795/4A cells were transferrin receptor (CD71)-positive, representing proliferating cells. Presumably as a consequence of PHA/IL-2 stimu- lation all 1795/4A cells expressed HLA-DR, whereas only a minor subpopulation of 1795 was HLA-DR- positive. The T cells of 1795 and 1795/4A differed further with respect to their CD2 antigen expression. The entire cell population of 1795 was positive for OKT11 (CD2), whereas the IL-2-dependent cell line 1795/4A showed only 60% reactivity with OKTl l .

Cytogenetic analysis The karyotype of the fresh tumor cell suspension

1795 has been published before [6, 32]. The mono- nuclear cells were derived from peripheral blood and revealed complex chromosomal aberrations with less than 10% normal cells. The karyotype was described as 44, X, - Y , -20 , 4 q - , 6 p - , 14q- , 19p+, 20q+, 22q- . After G-banding the structural changes were interpreted as reciprocal translocation t(4;20)(q13;q12), translocation t(6;19;22) (p l l ;p l3 ; q13), and a breakpoint in band 14qll due to a small interstitial deletion del l4(ql l ;ql3) , and a second

TABLE 1. SURFACE PHENOTYPE OF THE PARENTAL TUMOR CELL LINE (1975) AND THE ESTABLISHED CELL LINE (1795/

4A)

Monoclonal antibodies (mAb) Reactivity with mAb in % of cells 1795 1795/4A

CD1 (T6) 0 0 CD5 (T1) 97 98 CD7 (3A1) 97 97 CD2 (Tll) 98 58 CD3 (Leu4) 97 95 CD8 (Leu2A) 97 0 CD4 (Leu3A) 64 94 CD25 (TAC) 68 67 CD10 (CALLA) 0 0 CD71 (T9) 25 49 HLA-DR 27 96 IgG, A, M, D, K, X 0 0 CD20 (Leu16) 2 0 CD19 (Leul2) 0 0

interstitial deletion del14(q21;q24). Cell line 1795/ 4A exhibited comparatively few chromosomal changes. After long-term cell culture and repeated freezing and thawing a 45, XY, l l q + , - 22 karyotype with a reciprocal translocation t(14;18)(q11;q23) was described (Fig. 2). The nature of the l l q + remained unknown. This translocation is characterized by two distinct bands. There seemed to be no clear relation- ship of this translocation to chromosomal regions related to T-cell receptor genes. The translocation t(14;18) was detected in all 30 metaphases studied. The karyotype remained stable throughout the cul- ture period when repeatedly tested.

Restriction fragment analyses TCR-fl and -y gene rearrangements. In order to

determine the clonality of the fresh tumor cells 1795 and cell line 1795/4A, we compared the TCR-fl and TCR-7 rearrangement patterns of 1795 and 1795/4A. In each instance the Cfl-alleles exhibited a rearranged configuration (Fig. 3). The DNA of 1795 showed a rearrangement of Cfll when digested with EcoR1 and hybridized with a C/3-probe, and an additional rearrangement of Cff2, as shown by the Hindll digest. Both fragments comigrate in the BamH1 blot. Cell line 1795/4A exhibited a biallelic deletion of Cfll with EcoR1, and a rearranged Cfl2 fragment when digested with Hindlll, which recognizes rearrange- ments to the Cff2 locus [33]. A BamHl digest showed two rearranged fragments, indicating that both Cfl2 alleles have been rearranged. These two fragments comigrate in the Hindlll blot.

The clonal rearrangements were confirmed when EcoR1 and Kpnl digested DNA of 1795 and 1795/4A

684 M. UPPENKAMP et al.

was hybridized with a J71 probe, which recognizes the highly homologous Jy1.3 and J72.3 region genes [23] (Fig. 4). Tumor cells 1795 revealed a germline configuration with EcoRl of 3.2kb and 1.5kb, corresponding to J72 and Jy1 respectively. The rearrangement was detected when 1795-DNA was digested with Kpnl, indicating that these cells utilized a Jy region different from Jy1.3 and J72.3 [34]. Cell line 1795/4A exhibited a clonally rearranged EcoR1- and Kpnl-fragment of 0.6 and 1.9 kb, respectively.

The analysis of the TCR-/3 and TCR-7 genes by the Southern-blot technique revealed clonal rearrangements. No additional non-germline frag- ments were detected. Although 1795/4A was derived from the cell suspension 1795 by PHA and continuous IL-2-stimulation, no comigration of rearranged restriction fragments was observed.

TCR-6 gene rearrangements. We next analyzed the TCR-6 gene locus, which is located approximately 85 kb upstream of the constant Co~ region within the Vcr and Ja" gene segments on chromosome 14qll [15, 25, 26]. The TCR-6 gene is usually deleted in o~- /3 expressing T cells [26, 35]. We hybridized blots of EcoR1 and Hindlll digested DNA with a C6 region probe (data not shown). Placenta DNA served as germline control. 1795 has deleted both C6 regions, whereas 1795/4A exhibited a germline configuration with a half-intensity signal, suggesting that a mono- allelic rearrangement has occurred. From these find- ings and the phenotypic analysis it is concluded that 1795 and cell line 1795/4A have functionally rearranged one TCR-cr allele, which codes for the expression of the To:-fl receptor on the cell surface membrane.

Further restriction fragment analysis of the TCR- 6 gene locus with a J61 probe, which is selectively utilized in humans [25,36], revealed monoallelic rearrangements of 7.4 kb (EcoR1), 10.5 kb (BamHl), and 18 kb (Hindlll) in cell line 1795/4A (Fig. 5). The fresh tumor material 1795 showed a biallelic deletion of the J6 gene as was expected from the C6 analysis. Because of the unusual finding of a J6 rearrangement in a mature T-cell line with restriction fragments of rather large size (Hindlll fragment), we investigated, whether an incomplete TCR-6 rearrangement has occurred. The DNA was digested with the restriction enzyme Xbal, which encompasses the J61 segment gene and is suitable for detecting D6-J61 joinings, when hybridized with a J61 probe [37]. J61 was found in germline configuration in line 1795/4A (data not shown). Thus no D6--J61 joining has happened.

The J61 rearrangements may therefore represent joinings of D63 to a region further upstream, possibly another D6 or to a Vo:/6 region gene. Rehybrid- ization of EcoR1 and Hindlll blots with a V61-probe

showed a biallelic deletion in 1795 confirming the complete deletion of the TCR-6 gene locus. Cell line 1795/4A however displayed V61 germline fragments of half intensity signal, suggesting that neither a V61- (D)-J61 rearrangement nor a recombination of J61 to a V-region 5' of V61 has occurred on this allele. The second allele was deleted (Fig. 6).

In order to confine the J61 rearrangement in cell line 1795/4A we hybridized EcoR1 and BamH1 digested DNA with the genomic probe 5DHR7, which recognizes an EcoR1-Hindlll fragment between D62 and D63 (Fig. 1). Cell line 1795/4A showed a biallelic deletion of this region. Thus the TCR-6 rearrangement occurred upstream of J61 but downstream of V61.

We also investigated the putative breakpoint on chromosome 18q23. One gene that maps to the chromosomal region of interest codes for the myelin basic protein (MBP) [28, 29, 38]. Restriction frag- ment analysis with EcoR1, BamIql and Hindlll of 1795/4A-DNA with MBP probes revealed only germline fragments (data not shown). Thus, this gene seems not to be involved in the chromosomal trans- location t(14;18)(q11;q23).

DISCUSSION

Cell line 1795/4A is a newly established IL-2 dependent, permanently growing T-cell line from a patient with AT. The phenotype and morphology of the fresh tumor cells, termed 1795, were found to be consistent with the diagnosis of a chronic T-cell leukemia (T-CLL), as reported [6, 30, 32, 39]. Cell line 1795/4A displayed a somewhat more mature phenotype, but was clearly not derived from the major T-cell clone. It differed from the fresh tumor cells with respect to restriction fragment analysis and is characterized by the chromosomal translocation t(14;18). These cells are considered a minor T-cell clone within the fresh tumor cell suspension that remained undetected in the patient. This finding reflects the heterogeneity of the tumor with at least two tumor cell clones. The fact that nearly all (> 90%) lymphocytes exhibit the same pathological karyotype and that 97% express CD3, CD8 and CD4 +- antigens, makes it conceivable that the number of normal T cells is very low and that not all T cells in the patient were abnormal at this stage.

The TCR-6 gene is frequently rearranged in human precursor T-cell neoplasma that potentially express the y-6 TCR on the surface membrane, but is deleted by TCR-cr rearrangements in or-/3 expressing T cells [12, 17, 26, 27, 35, 37]. Studies of the TCR-6 gene in these tumors and human peripheral blood y-6 bear- ing T cells displayed a limited repertoire of V6 genes.

2

1 2 3

6 7 8

4 5

i( t l ,. 9 10 11 12

ii ~" It 13 14 15

at t( 19 2 0

,t :( 'I 16 17 18

2t 22

| X Y

3

12 k b I -

4.0 k b -

Q . " ~ T -

I ! I

~ ~ ~ii~i~i ~ ~ ~i

I I I

x

7.4 kb . . . . . -

3.4 ~

m < G ,q" o tn In

C o o~ o~ a r,,, r,..

I ! I

24 kb . I -

E x

FIG. 2. Karyotype of the established cell line 1795/4A with 45, XY. l l q + , - 2 2 and a reciprocal translocation

t(14; 18)(q 11 ;q23).

FIG. 3. Southern-blot analysis of the parental tumor cell line 1795 and T-cell line 1795/4A. DNA was digested with the restriction enzymes EcoR1 (A), Hindlll (B), and BamHl (C) and hybridized with a c/3 region probe. Germline fragments (placenta) are 12 and 4.0 kb for EcoR1; 7.4 and

3.4 kb for Hindlll; 24 kb for BamHl as indicated.

685

~1 I ~

A ,-

I i i

3.2 I r ~

1 . 5 k b - kb

FIG. 4. Southern-blot analysis of the parent tumor cell line 1795 and the cell line 1795/4A. DNA was digested with EcoRl (A) and Kpnl (B) and hybridized with a J~, region probe. Germline fragments (placenta) are 3.2 (Jr2) and 1.5 (Jy1) kb for EcoRl and 16 and 9 kb for Kpnl as shown.

Rearrangements are marked by arrows.

686

5

A

I

m

u

I !

I 6.6

B

kb

C

6

A

t

7.4 0

3.2 kb--- ' . ~) Q

FIG. 5. Southern-blot analysis of DNA from cell line 1795 and 1795/4A. The DNA was digested with the restriction enzymes EcoR1 (A), BamH1 (B), and Hindlll (C) and hybridized with a J6 region probe. Germline fragment sizes (placenta) are indicated. The rearranged fragments are

marked by an arrow.

FIG. 6. Restriction fragment analysis of DNA from cell line 1795 and 1795/4A. The DNA was digested with EcoRl (A) and Hindlll (B) and hybridized with a V61 region probe. Placenta DNA served as germline control. Germline frag- ments are 3.2 kb for EcoRl and 7.4 kb for Hindlll as indi-

cated.

687

Molecular analysis of an ataxia telangiectasia T-cell clone 689

To date 3-6 V6 genes have been described in humans [17, 37, 40]. Rearrangements of 3 major V6 sub- families (V61, V62, and V63) have been identified within precursor T-cell ALL, lymphoid precursor leukemias, and lymphoblastic lymphomas [17, 18, 37]. As a result of the limited diversity of the TCR-6 gene, distinct types of TCR-6 rearrangements have been reported using a J61 probe. Accordingly V61-3(D)-J61 recombinations can be identified by their restricted rearrangement pattern [17, 18, 37, 41]. Southern-blot analysis of line 1795/4A, however, yielded monoallelic J61 rearrangements of atypical size that did not resemble the restriction pattern of the predominantly used V6-genes. In fact, hybrid- ization to a V61-specific probe showed germline con- figuration of one allele. The second TCR-6 allele was deleted, probably due to a functional TCR-o~ rearrangement. We therefore speculate that no major V6-gene is involved in the rearrangement described here.

Although an incomplete V-D joining with a new or infrequently utilized V6-gene could not be excluded completely, we propose that a chromosomal break- age occurred 5' of J61, deleting the DNA sequence that retains the EcoR1-Hindlll fragment upstream of D63. We imply that the breakpoint is located on a small Xbal-Hindll! fragment containing the D63 segment, because the restriction analysis of J61 revealed germline configuration when digested with Xbal, but exhibited a rearrangement in the Hindlll- digest. Thus the TCR-6 locus seems to be split between V6 and J6 segments, recombining the J61- C6 region to a yet undescribed region on chro- mosome 18q23.

Chromosomal translocations are non-random events in patients with AT and are probably due to a defective recombinase action and to impaired repair mechanisms during T-cell ontogeny, which is defec- tive in AT-patients [4, 42,43]. They frequently involve region 14qll (TCRcr/6) [4,5,7-10]. The deletion of the TCR-6 gene for functional TCR-o: expression and the large number of JR segments spanning over 100 kb are prone to errors in TCR gene rearrangements, leading to translocations [26, 35, 44, 45].

Although chromosomal translocations involving 14qll may not necessarily cause malignant trans- formation, they seem to be associated with a pro- liferative advantage [46]. T-cell clone 1795/4A, however, showed no growth advantage in vivo, but a continuous proliferation rate upon IL-2 stimulation in vitro. It therefore may be regarded as a pre- malignant clone that has to accumulate further mol- ecular aberrations in order to become malignant.

This cell line provides a unique model for studying

the heterogeneity of a tumor cell population with the existence of distinct cell clones on a molecular genetic base. Strong evidence is provided that the chromo- somal breakage in cell line 1795/4A occurred within the TCR-6 locus. Cloning and sequencing analyses are under way to pin point the exact breakpoint on chromosome 14 and to identify the genes involved in the breakpoint region of chromosome 18q23. This chromosomal region could conceal a new oncogene.

Acknowledgements--We are grateful to Drs T. Mak, T. Rabbitts, E. Loh, M. Krangel, and D. Cohen for providing molecular probes. We thank I. Uppenkamp for excellent technical assistance.

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690 M. UPPENKAMP et al.

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