Establishment and Characteristics of a T-cell Acute Lymphoblastic Leukemia Cell Line, JK-T1, with a Chromosomal Translocation Between 8q24 and 14q13 Mitsuyoshi Urashima, Hideaki Iyori, Kouji Fujisawa, Yasutaka Hoshi, Jun-ichi Akatsuka, and Kihei Maekawa

ABSTRACT: A human leukemia cell line, JK-T1, was established from the bone marrow of a lO-yearold boy with T-cell acute lymphoblastic leukemia. The origin of the leukemic cell line, JK-T1, was demonstrated by its chromosomal and immunologic similarity to the patient's fresh leukemic cells.

Karyotypic analysis revealed 46,XY, del(6)(q?),t(8;14)(q24;q13),der(9)t(9;?)(q34;?). In JK-T1, neither rearrangement nor amplification of the c-myc gene was observed apparently because the breakpoint of chromosome 14 was not q l l but q13. JK-T1 was independent of interleukin 2 (IL-2) because of little production of IL-2, little IL-2 receptor (CD25) on the surface, and no response to exogenous IL-2. JK-T1 had lymphocyte function associated antigen-1 (LFA-1) (CD11a, CD18) on its surface and could adhere to the hematologic stromal layer. These characteristics of JK-T1 cell line are considered to be be useful not only for evaluating the role of t(8;14) but also in studying the adhesion molecules of leukemia.

INTRODUCTION The relationship between chromosomal abnormalities and oncogenes has been thoroughly investigated in leukemias. One of the activation m e c h a n i s m s that impairs oncogene expression in leukemic cells consists of chromosomal translocation. A reciprocal translocation t(8;14)(q24;q32) in Burkitt l y m p h o m a is one of the w e l l - k n o w n chromosomal abnormalities [1], and the other translocations, t(2;8)(p12;q24) [2] and t(8;22)(q24;q11) [3], have also been reported in this disease. The locus 8q24 is k n o w n to encode the oncogene c-myc, while 14q32, 2p12, and 2 2 q l l encode the genes for the i m m u n o g l o b u l i n heavy-chain,the K-chain, and the k-chain, respectively. These translocations alter the transcriptional regulation of c-myc, w h i c h is believed to play an important role in the pathogenesis of this tumor [4]. W h e n T-cell leukemia cases were c o m p a r e d with Bcell cases, significantly more chromosomal translocations occurred near T-cell receptor (TcR) gene loci in the former than in the latter [5]. Translocation t(8;14)(q24;q11) is one of the w e l l - k n o w n chromosomal abnormalities that occurs in patients with T-cell acute l y m p h o b l a s t i c leukemia (TALL) [6-8]; this translocation also occurs in other cell lines [9, 10]. In T-ALL, t(7;12)(q32-34;q24) has also been re-

From the Department of Pediatrics, Jikei University School of Medicine, Tokyo, Japan. Address reprint requests to: Dr. Mitsuyoshi Urashima, Department of Pediatrics, Jikei School of Medicine, 3-25-8 Nishi-shinbashci, Minato-ku, Tokyo 105, Japan. Received April 16, 1992; accepted June 5, 1992. 86 Cancer Genet Cytogenet 64:86-90 (1992) 0165-4608/92/$05.00

ported [11]. The locus 1 4 q l l is k n o w n to encode the TcRc~and y-chains [8]; the locus 7q32-q36 encodes the TcR flchain [11]. These loci are translocated to the 3' side of the c-myc-coding exons on c h r o m o s o m e 8 [12-14]. Therefore, this type of translocation present in T-ALL m a y be analogous to the variant translocation of Burkitt l y m p h o m a , suggesting that the TcR gene plays a role similar to that of the i m m u n o g l o b u l i n gene in Burkitt l y m p h o m a . We also report the establishment of a n e w T-ALL cell line, JK-T1, with translocation between 8q24 and 14q13. It is intriguing that the breakpoint was not 1 4 q l l but 14q13. The characteristics of the cell line were studied.

PATIENT AND METHODS Case Report The patient was a 10-year-old boy with c o m p l a i n t s of fever, vomiting, and diarrhea. He was pale, w i t h bleeding tendencies and hepatosplenomegaly. The white blood cell count was 578,000//~1, with 94% lymphoblasts. Hemoglobin was 10.3 g/dl; the platelet count was 67000/~1. A s p i r a t i o n of the bone marrow showed that the m a r r o w contained more than 90% lymphoblasts. L2 m o r p h o l o g y was observed for the leukemic cells according to the F r e n c h - A m e r i c a n - B r i t i s h (FAB) classification, and the cells were m y e l o p e r o x i d a s e (MPO)-negative, periodic-acid Schiff (PAS)-negative, anaphthylbutyrate esterase (NBE)-negative, and n a p h t h o l AS-D chloroacetate esterase (CAE)-negative. After leukapheresis, i n d u c t i o n of r e m i s s i o n was initiated with a regimen of prednisone, vincristine, adriamycin, and L-asparaginase. Complete hematological remission © 1992 Elsevier Science Publishing Co.. Inc. 655 Avenue of the Americas, New York, NY 10010

JK-T1 Establishment

Table 1

87

Reactivity of original leukemic cells JK-T1 cell line with m o n o c l o n a l antibodies

CD

Original leukemic cells (%)

JK-T1 cells (%)

2 3 5 7 11 a 18 38

90.5 58.9 85.1 53.2 ----

98.0 90.8 94.6 70.0 92.4 91.3 97.4

was not achieved; the patient d i e d of intracranial hemorrhage 3 months later.

Cell Culture Bone marrow was collected from the patient in a heparinized syringe before chemotherapy, layered over FicollH y p a q u e (specific gravity 1.078), and centrifuged at 400 g for 30 minutes. The band of m o n o n u c l e a r cells at the interface was removed and w a s h e d three times with phosphatebuffered saline, and r e s u s p e n d e d until a concentration of 1.0 x 106 cells/ml was achieved in RPM! 1640 culture m e d i u m . This culture m e d i u m consisted of heat-inactivated 10% fetal calf serum (Gibco), and antibiotics (penicil-

lin G,100 U/ml; streptomycin, 0.1 mg/ml; a n d a m p h o t e r i c i n B 0.25,/zg/ml; Sigma). Flasks containing 10 ml of the med i u m with the final cell concentration of 1.0 x 107 cells/ 10 ml were prepared and i n c u b a t e d in h u m i d i f i e d air with 5% CO 2 and 5% 02 at 37°C. Every 2 - 7 days, 5 ml of the growth m e d i u m was r e p l a c e d by an equal v o l u m e of fresh growth m e d i u m to "feed" the culture. The cells were resusp e n d e d in flesh growth m e d i u m every month. Colony assay was performed using a m e t h y l c e l l u l o s e s e m i s o l i d culture (1.0 × 10 s cells, 2% m e t h y l c e l l u l o s e 0.2 ml; m e d i u m , 0.8 ml). Culture cells were observed using phase contrast microscope and were counted with a hemacytometer.

Morphology and Cytochemical Staining Cells s e d i m e n t e d in a cytocentrifnge were stained using the standard procedures [15] for Wright, MPO, PAS, NBE, and CAE stains. Terminal d e o x y n u c l e o t i d e transferase was d e t e r m i n e d by standard techniques [16]. Surface markers (CD2, 3, 5, 7, 11a, 18, 38) were analyzed as r e p o r t e d previously [17].

Chromosomal Analysis Fifty b a n d e d metaphases from both the p a t i e n t ' s bone marrow blood and the JK-T1 cell line were a n a l y z e d by the Qbanding technique, as reported previously.

Figure 1 Representative karyotype of JK-T1 cell line: 46,XY,del(6)(q?),t(8;14)(q24;q13),der(9)t(9;?)(q34;?).

88

M. Urashima et al.

--i~ 0

D_ii}lii112 "

12

c-myc I oncogene

Figure 2

] TcR ~ chain

translocation between 8q24 and 14q 13

Diagramof the breakpoint and translocation between 8q24 and 14q13.

Interleukin-2 Measurement Production of IL-2 by the JK-T1 cells (initial concentration at 1.0 × 105 cells/ml) was measured by analyzing its filtrate using radioimmunoassay as previously mentioned [18] on the 10th day of culture. Reactivity to IL-2 was measured by adding IL-2 to the JK-T1 conditioned culture medium (10 ml}. The concentrations of IL-2 were 0, 10, 50,100, and 200 /~g//~l. Cell numbers at each concentration were counted on day 7 of cultivation and compared.

DNA Analysis Genomic DNA was extracted from control cells and from monoclonal expansions of leukemic cells, and then was digested with restriction endonucleases (BamHI, EcoRV) known to demonstrate both the rearranged and germ-line configurations of the T-cR C/31 gene. Such digested DNA was fractionated by size using agarose gel-electrophoresis, transferred to diazobenzyloxymethyl paper, hybridized with nick-translated 32p-DNA probes, and visualized on autoradiograms [19]. C-myc gene was analyzed according to the method previously described [20]. The DNA was digested with restriction endonucleases (EcoRI, SacI, Bg III), and a 32p c-myc DNA probe was utilized to detect amplification and rearrangement. The human placenta was used as the negative control, and HL60 cell line was used as the positive control. The c-abl gene was also analyzed according to the method previously described [21]. The

DNA was digested with restriction endonucleases (EcoRI, Bam HI, Hind III), and a 32p c-abl DNA probe was utilized to detect amplification and rearrangement. The human placenta was used as a negative control, and K562 cell line was used as a positive control.

Preparation of Bone Marrow Stromal Layer Normal bone marrow mononuclear cells were obtained from healthy donors for allogenic bone marrow transplantation. The bone marrow cells were separated and cultured as described in cell culture. Adherent stromal cells covered the bottom of flask, and nonadherent cells were rarely found after 8 weeks. At 12 weeks of culture, the JK-T1 cells were added on the adherent stromal cell layer (concentration; 1.0 × 1 0 6 cells/ml) and observed with phase-contrast microscope. RESULTS Growth Properties Cultured cells grew rapidly without the development of any stromal cells from the initiation culture. The cell doubling time was about 36 hours. These cells have been maintained for at least 4 months. Neither feeder layer nor growth factors were required for initiation or maintenance of the culture. In the methylcellulose culture system, the cells formed small clusters after 2 weeks of culture.

JK-T1 Establishment

89

12.0Kb 7.0Kb R

EcoRV Bam H I Figure 3 TcR C/31-chain gene rearrangement. EcoRV- and BamHI-digested DNA isolated from JK-T1 cells displayed clear monoclonal or oligoclonal rearrangement of the Cbl allele, visible as distinct bands at R.

Similarities to the Patient's Leukemic Cells Immunologic data comparing the patient's original leukemic cells and the JK-T1 cell line are shown in Table 1. The same T-cell immunologic characteristics (CD2, 3, 5, 7) were recognized in both the patient's original leukemic cells and JK-T1. All of the cytochemical staining (PAS, MPO, NBE, CAE, TdT) were negative both in original leukemic cells and in JK-T1 cells. Characteristics of the JK-T1 Cell Line The karyotype of the JKIT1 cell line at 4 months of cultivation is shown in Figure 1, and a diagram of breakpoints and translocations was shown in Figure 2. The karyotype was 46,XY,del(6)(q21),t(8;14)(q24;q13)der(9)t(9;?)(q34;?). TcR Cbl chain gene rearrangement is shown in Figure 3. The EcoRV and Bam HI-digested DNA isolated from the JK-T1 cells displayed clear monoclonal or oligoclonal rearrangement of the Cfll allele, visible as distinct bands at R. Neither amplification nor rearrangement of the c-myc gene and c-abl gene was detected. (The data was not shown.) JK-T1 did not react with CD25 bound to the IL-2 receptor a-chain. IL-2 was not detected in the culture me° dium conditioned by JK-T1, and there was no change in growth properties when IL-2 was added to the medium. JK-T1 cells were adherent to the prepared bone marrow stromal layer. CD11a and CD 18 were highly expressed. DISCUSSION Continuous proliferation of T-ALL cells was observed for at least 4 months. The maintenance of the same T-cell chromosomal abnormalities and surface markers during culture demonstrated that JK-T1 was derived from leukemic cells. JK-T1 exhibited monoclonality with respect to TcR-~ chain gene rearrangement patterns. Therefore, JK-T1 was considered to be established as a T-ALL cell line. JK-T1 is intriguing with regard to the specific chromosomal abnormalities near the portion of TcR and c-myc genes, IL-2-independent growth, and its adhesion properties. In T-ALL with t(8;14)(q24 ;ql 1), it has been observed that locus 1 4 q l l that encodes the TcR a- and 7ichains were

translocated to locus 8q24, which is on the 3' side of the c-myc gene; this leads to deregulation of the c-myc oncogene [12-14]. However, in the JK-T1 cell line, the breakpoint of chromosome 14 was not q l l but q13. So, the TcR a- and 7-chain gene did not appear to have been transferred to chromosome 8. That is why neither amplification nor rearrangement of c-myc was detected. Oppositely, amplification and rearrangement of c-myc gene might need for the involvement of TcR gene. Deletion of 6q chromosome was also recognized with T-ALL [22]. The c-abl gene is located on chromosome 9, and rearrangement of c-abl gene is often observed in chronic myelogenous leukemia [21]. However, in JK-T1 cell line, rearrangement of c-abl gene was not recognized in spite of 9q(+). Autocrine growth factors [23], such as IL-2 [24], were also considered to play a role in the pathogenesis of T-ALL. JK-T1, however, was found not to be dependent on IL-2, because there was little IL-2 production, little IL-2 receptor expression on the cell surface, and no response to IL-2. The pathogeneses of T-All are various. JK-T1 demonstrated adhesive properties and possessed lymphocytes function associated antigen (LFA-1) (CD11a and CD18). In T-All with high leukocyte count, LFA-1 is high [25]. But the level of intercellular adhesion molecule° 1 (ICAM-1) (CD54) was low. They did not aggregate with one another enough. However, the cells adhered to the bone marrow stromal cell layer. Pals et al. [26] pointed out that the cells that had LFA-1 could react to endotheial venules. Damle and Doyle [27] described that T lymphocytes with CD3 molecules induces enhancement in vascular endothelial permeability with participation of intercellular adhesion pathways. Thus, the adhesion molecules of JK-T1 may be partially responsible for intracranial bleeding. In conclusion, the JK-T1 cell line was newly established as a T-ALL cell line. It is intriguing in respect to its specific chromosomal aberration, IL-2 independent growth, and adhesion properties.

REFERENCES 1. Klein G, Klein E (1985): Evolution of tumors and the impact of molecular biology. Nature 315:190-195. 2. Adolph S, Hameister H, Henglein B, Lipp M, Hartl P, Baas F,

90

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

M. U r a s h i m a et al. Lenoir G, Bornkamm GW (1989): t(2;8) variant translocation in Burkitt's lymphoma: mapping of chromosomal breakpoints by in situ hybridization. Int J Cancer 44:261-265. Popescu NC, Dahlberg JE, Ablashi DV, Monastier M, Bona CA, DiPaolo JA, Hooper WC, Swan DC (1990): Oncogene expression and immunoglobulin synthesis in a North American Burkitt (NAB-2) lymphoma cell line with a 8;22 chromosome translocation. Oncogene Res 5:295-303. Zajac-kaye M, Gelmann EP, Levens D (1988): A point mutation in the c-myc locus of a Burkitt lymphoma abolishes binding of a nuclear protein. Science 240:1776-1780. Raimondi SC, Behm FG, Roberson PK, Pui CH, Rivera GK, Murphy SB, Williams DL (1988): Cytogenetics of childhood Tcell leukemia. Blood 72:1560-1566. Hayashi Y, Yamamoto K (1986): T-cell acute lymphoblastic leukemias with a t(8;14) possibly involving a c-myc locus and T-cell-receptor alpha chain genes. New Eng J Med 314:650-651. Inaba T, Murakami S, Oku N, Itoh K, Ura Y, Nkanishi S, Shimazaki C, Nishino A, Nakazawa M, Fujita N, Nishigaki H, Taniwaki M, Misawa S (1990): Translocation between chromosomes 8q24 and 1 4 q l l in T-cell acute lymphoblastic leukemia. Cancer Genet Cytogenet 49:69-74. McKeithan TW, Shima EA, LeBeau MM, Minowada J, Rowley JD, Diaz MO (1986): Molecular cloning of the breakpoint junction of a h u m a n chromosomal 8;14 translocation involving the T-cell receptor alpha-chain gene and sequence on the 3' side of MYC. Proc Natl Acad Sci USA 83:6636-6640. Minowada J, Sagawa K, Trowbridge IA, Kung PD, Goldstein G (1982): Marker profiles of 55 h u m a n leukemia-lymphoma cell lines. In: Malignant Lymphomas, SA Rosenberg, HS Kaplan, eds. Academic, New York, pp. 53-74. Mathieu-Mahul D, Sigauz F, Chen A, Bernheim A, Mauchauffe M, Daniel MT, Berger R, Larsen CJ (1986): A t(8;14)(q24;q11) translocation in a T-cell leukemia (L1-ALL) with c-myc and TcR-alpha chain locus rearrangements. Int J Cancer 38:835-840. Kees UR, Lukeis R, Ford J, Garson M (1989): Establishment and characteristics of a childhood T-cell acute lymphoblastic leukemia cell line, PER-255, with chromosome abnormalities involving 7q32-34 in association with T-cell receptor beta gene rearrangement. Blood 74:369-373. Croce CM, Isobe M, Palumbo A, Puck J, Ming J, Tweardy D, Erikson J (1985): Gene for alpha-chain of h u m a n T-cell receptor: Location on chromosome 14 region involved in T-cell neoplasms. Science 227:1044-1047. Erikson J, Finger L, Sun L, Rushdi A, Nishikura K, Minowada J, Finan J, Emanuel BS, Nowell PC, Croce CM (1986): Deregulation of c-myc by translocation of the alpha-locus of the T-cell receptor in T-cell leukemia. Science 232:884-886.

14. Erikson J, Williams DL, Finan J, Nowell PC, Croce CM (1985): Locus of the alpha-chain of the T-cell receptor is split by chromosome translocation in T-cell leukemias. Science 229:784-786. 15. Loftier H (1971): Zytochemische Klassifizierung der Leukosen bei Erwachsenen. Acta Histochem 9:181-184. 16. Hoffbrand AW, Janossy G (1981): Enzyme and membrane markers in leukemia: Recent developments. J Clin Pathol 34:254-262. 17. Foon AK, Todd RF (1986): Immunologic classification of leukemia and lymphoma. Blood 68:1-37. 18. Gillis S, Gjerset R, Yu A, Haas M (1990): Establishment of continuous cultures of T-cell acute lymphoblastic leukemia cells at diagnosis. Cancer Res 50:10-14. 19. Isobe M, Sadamori N, Russo G, Shimizu S, Yamamori S, Itoyama T, Yamada Y, Ikeda S, Ichimaru M, Kagan J, Croce CM (1990): Rearrangement in the h u m a n T-cell-receptor a-chain locus in patients with adult T-cell leukemia carrying translocation involving chromosome 14q11. Cancer Res 50:6171-6175. 20. Wong AJ, Ruppert JM, Eggleston J, Hamilton SR, Baylin SR, Vogelstein B (1986): Gene amplification of c-myc and N-myc in small cell caricinoma of the lung. Science 233:461-462. 21. Morris SW, Daniel L, Ahmed CM, Elias A, Lebowitz P (1990): Relationship of bcr breakpoint to chronic phase duration, survival, and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase. Blood 75:2035-2041. 22. Baletta C, Pelicci PG, Kenyon LC, Smith SD, Dalla-Favera D (1987): Relationship between the c-myc locus and the 6qchromosome aberration in leukemias and lymphomas. Science 23:1064-1067. 23. Vittenbogaart CH, Nishanian PG, Anisman DJ, Erikson TK, Fahey JK (1986): Leukemia derived growth factors produced by h u m a n malignant T-lymphoid cell lines. Cancer Res 46:1219-1223. 24. Duprez V, Lenoir G, Dautry VA (1985): Autocrine growth stimulation of a h u m a n T-cell lymphoma line by interleuken 2. Proc Natl Acad Sci USA 82:6932-6936. 25. Schirren CA, Volpel H, Meuer SC (1992) Adhesion molecules on freshly recovered T leukemias promote tumor-derived lympholysis. Blood 179:138-143. 26. Pals ST, Horst E, Scheper RJ, Meijer CTLM (1989): Mechanisms of h u m a n lymphocyte migration and their role in the pathogenesis of disease. Immunol Rev 108:111-133. 27. Damle NK, Doyle LV (1990): Stimulation of cloned h u m a n T lymphocytes via the CD3 or CD28 molecules induced enhancement in vascular endothelial permeability to macromolecules with participation of type-1 and type-2 intercellular adhesion pathways. Eur J Immunol 20:1995-2003.

Establishment and characteristics of a T-cell acute lymphoblastic leukemia cell line, JK-T1, with a chromosomal translocation between 8q24 and 14q13.

A human leukemia cell line, JK-T1, was established from the bone marrow of a 10-year-old boy with T-cell acute lymphoblastic leukemia. The origin of t...
501KB Sizes 0 Downloads 0 Views