© 1990 S. Karger AG, Basel 0001-5792/90/0843-0156 S 2.75/0
Acta Haematol 1990;84:156-161
Establishment and Characterization of Acute B-Cell Lymphocytic Leukemia Cell Line Showing (8;14) and (14;18) Chromosome Translocation1 Hiromitsu Matsuzakï', Hiroyuki Hata', Norio A sow', Hitoshi Suzushimaa , Yoshitaka Akalwshï', Minoru Yoshidaa , Shouichi Nagakuraa, Toshinori Ishiih, Isao SanadaK, Kiyoshi Takatsukia a Second Department of Internal Medicine, Kumamoto University Medical School, b Department of Medical Technology, College of Medical Science, Kumamoto University, and c Department of Internal Medicine, Kumamoto National Hospital, Kumamoto, Japan
Key Words. Acute lymphocytic leukemia ■Bcl-2 • Cell line ■Myc • Translocation
Introduction Specific chromosomal translocations are consis tently found in some hematological malignancies. For example, a t(8; 14) (q24;q32) translocation is observed in 70-80% of Burkitt’s lymphomas [1]. Dalla-Favera et al. [2] have shown that the c-myc oncogene, which is located on band q24 of human chromosome 8 [2, 3], translocates to the immunoglobulin heavy chain locus on chromosome 14, which is implicated in Burkitt’s lymphoma with the 8; 14 translocation [4], In a minor proportion of Burkitt’s lymphomas, which exhibit a variant t(2;8) or t(8;22) translocation, the c-myc onco gene is placed in proximity to the k or X light chain constant (CKor C\) regions, respectively [5, 6], The consequence of this chromosome rearrangement is a
1 Supported in part by a Grant-in-Aid for Cancer Research (61-2) from the Ministry of Health and Welfare, and a grant (63770903) from the Ministry of Education, Science and Culture of Japan.
deregulation of the involved c-myc gene, so that it is transcribed constitutively at an elevated level [5-7], and the pathogenesis of this high-grade B-cell malig nancy. Another chromosomal translocation, t( 14; 18) (q32;q21), involving the immunoglobulin heavy chain locus on chromosome 14q32, is found in follicular lymphoma [8-10], Tsujimoto et al. [11] postulated a putative protooncogene, bcl-2, which is normally lo cated on band q21 of chromosome 18 and may be moved into close proximity to a rearranged heavy chain locus in band 14q32. By using a probe for the joining (JH) segment of the immunoglobulin heavy chain locus, they and others have been able to identify a bcl-2 [11, 12]. They also reported that bcl-2 has been found to be rearranged in more than 80% of follicular lymphomas [13]. In this report, we have characterized a cell line, KHM-2B, which was derived from a patient with acute lymphoblastic leukemia, who carries both the 14; 18 and the 8; 14 translocations.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
Abstract. Cell line KHM-2B expressing two oncogene products, c-myc and bcl-2, was established from a pa tient with actute lymphocytic leukemia with an 8; 14 and 14; 18 chromosome translocation. Surface marker studies of the cell line showed that the cells were positive for HLA-DR, CALLA (CD10), B1 (CD20) and B4 (CD19), but negative forTl 1 (CD2). The fresh cells from peripheral blood of the patient had no surface immu noglobulins, whereas KHM-2B cells were positive for p-^type surface immunoglobulin. A cytogenetic analy sis of the cell line revealed two translocations, t (8; 14) (q24;q32) and t( 14; 18)(q32;q21). Rearrangement of the c-myc and bcl-2 genes was detected by Southern blot analysis of the KHM-2B DNA. Northern blot analysis revealed production of c-myc and bcl-2 mRNAs. These results indicated that two oncogenes were activated by two translocations to immunoglobulin genes.
157
ALL Cell Line with t(8; 14) and t( 14; 18)
Materials and Methods
Cell Culture Heparinized peripheral blood from the patient was layered on Ficoll-Conray (specific gravity 1.078) and was centrifuged at 400 g for 30 min. The interphase cells were collected and seeded into cul ture plates at approximately 106 cells/ml after washing with com plete medium. In primary culture and the early passages, RPMI 1640 medium containing 20% fetal calf serum was used, but when the cells began to grow steadily, the medium was changed to RPMI 1640 containing 10% fetal calf serum. The cells were maintained throughout under 5%CO; in humidified air at 37 °C. Immunological Marker Studies Measurement of the following surface markers was carried out: HLA-DR, CD2 (T il), CD3 (T3), CD4 (T4), CD8 (T8), CD10 (CALLA), CD19 (B4), CD20 (Bl) (Becton Dickinson, Mountain View, Calif.), and surface immunoglobulins (slg). Cytogenetics Cytogenetic studies of KHM-2B cells were performed using conventional trypsin-Giemsa chromosome banding techniques [14]. Karyotypes were arranged according to the criteria of the Paris Conference [15]. Southern Blot Analysis DNA was analyzed as described previously [16]. Briefly, 10 pg of high molecular weight DNA was digested with appropriate re striction enzymes, electrophoresed in 0.7% agarose gel and trans ferred to nitrocellulose filters. After being prehybridized at 42°C in plastic sealed bags containing 5 x standard saline citrate (SSC), 5 x Denhardt’s solution, 50% formamide, 20 mAf sodium phosphate buffer and heat-denatured salmon sperm DNA (200 pg/ml), filters were hybridized with a 32P-labeled c-DNA probe at 42°C, and washed 3 times at room temperature with 2xSSC/0.1% sodium dodecyl sulfate and then washed at 56“C with 0.1 x SSC/0.1% so dium dodecyl sulfate. Finally, filters were exposed to X-ray film with intensifying screens overnight at -70°C.
Fig. 1. Cytologic appearance of KHM-2B cells. KHM-2B cells had nuclei containing nucleoli and slightly basophilic cytoplasm with fine vacuoles (Wright-Giemsa stain).
Northern Blot Analysis Total cellular RNA was extracted using the guanidine thiocyanate/cesium chloride method as described elsewhere [17], Ten mi crograms of total RNA was denatured by heating at 60°C for 20 min in 50% (vol/vol) formamide and electrophoresed in 1% aga rose gel containing 2.2 M formaldehyde. After transferring the RNA to nitrocellulose filters, the filters were baked for 2 h at 80°C followed by hybridization, washing and autoradiography as in Southern blotting. DNA Probes The probes used in this study were a 2.5-kilobase (kb) £coRIBgl II fragment containing the Ig heavy chain joining region gene (provided by Dr. T. H. Rabbitts, Cambridge, UK) [18], a 1.8-kb C/al-EcoRI fragment containing the third exon of the human cmyc gene (purchased from ONCOR, Inc., Gaitherburg, Md.), and a 2.8-kb £coRI-Hifu/III fragment containing the bcl-2 gene (pur chased from ONCOR, Inc.).
Results Morphology o f Cultured Cells. The blast cells were collected and cultured from the patient’s peripheral blood before chemotherapy. At early passages, KHM-2B floated in the culture medium as single cells. At a later passage, some of them formed soft
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
Case Report Cell line KHM-2B was derived from the peripheral blood of a 62-year-old man, who was admitted to our hospital in June 1988 for fever, left abdominal pain, diarrhea and anorexia. Physical exami nation revealed only moderate hepatosplenomegaly but no lymph node swelling. Hematologic tests showed an increased leukocyte count (13,500/mm5) with 59% blast cells, a hemoglobin concentra tion of 7.4 g/dl, and a platelet count of 51,000/mm3. The nucleated cell count of bone marrow was 90,000/mm5 including 96.4% blast cells. Using Wright-Giemsa staining, blast cells showed a high nucleo-cytoplasmic ratio, with fine chromatin and several vacuoles in cytoplasm. Blast cells were negative for peroxidase and positive for periodic acid-Schiff stain. On the basis of blast cell morphology, the patient was suspected to have acute lymphoblastic leukemia type L3 (FAB classification). Clinical improvement was achieved transiently by intensive chemotherapy with daunomycin, vincris tine, L-asparaginase and prednisolone. However, increase of drugresistant blast cells and abdominal mass rapidly developed, and the patient died in September 1988. Autopsy revealed acute lympho cytic leukemia, with invasion into widespread lymph nodes, liver, spleen, intestine and peritoneum.
158
M a tsu zak i/H a ta/A so u /S u zu sh im a/A k ah o sh i/Y o sh id a/N ag ak u ra/Ish ii/S an ad a/T ak atsu k i
il
i f
i
2
3
l i 6
I I 7
H 8
» « 13
M I4
n
•
• 19
1
I f 4
1 f 9
• « 16
•
) 5
Il
U I2
11 17
1 * 18
) « XY
22
3
4
1 2
3
Fig. 2. Karyotype of the KHM-2B cells: 46, XY, t(8; 14) (q24;q32), t( 14; 18)(q32;q21), -21, + der(21)t(21; ?)(p 12; ?). As a result of the two reciprocal chromosomal translocations, the KHM-2B cells have two abnormal chro mosomes 14 (14q+), one rearranged chromo some 8 (8q-), and one abnormal chromosome 18(18q—).
4
kb '
»»
:
6.6-
1
l
9.4-
I I
1
2 3.1-
«
4.4-
bel 2
JH
clusters that continued to float. In Wright-Giemsastained smears, KHM-2B cells were morphologically similar to fresh cells, having nuclei containing nucle oli and slightly basophilic cytoplasm with vacuoles (fig- !)• Surface Markers. Fresh cells and KHM-2B cells were positive for HLA-DR, CD10 (CALLA), CD19 (B4), and CD20 (Bl). KHM-2B cells expressed p-A. type slg which was not detected in the freshly isolated cells (table 1). Karyotypic Analysis. Cytogenetic analysis of the
c-myc m
KHM-2B cells is of particular interest. All the cells of 15 metaphases analyzed were found to have t(8;14)(q24;q32) and t( 14; 18)(32;q21) (fig. 2). An addi tional chromosomal aberration such as -21, + der(21)t(21;?)(pl2;?) in figure 2, which was not clonal, was also detected. Southern Blot Analysis. Analysis of gene rearrange ment helped to determine the clonality of the cell line. Fresh lymphoblast cells from the patient and K.HM-2B cells showed the same rearranged bands of immunoglobulin, bcl-2 and c-myc genes (fig. 3). Both
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
2.3-
Fig. 3. Southern blot analysis of DNA from CEM (1), fresh lymphoblast cells (2), KHM-2B cells (3), and patient’s peripheral blood lymphocytes during complete remis sion (4) after digestion with EcoRI. The ni trocellulose filter was hybridized with bcl-2, J„, and c-myc probes. The arrows indicate the rearranged bands.
ALL Cell Line with t(8;14) and t( 14; 18)
159
samples, digested with £coRI, revealed rearranged JH fragments 6.2 and 10.2 kb in size. The size of the bcl-2 rearranged fragment was the same as the 6.2-kb J h rearranged band, which was also confirmed by Hind III digestion (data not shown). On the other hand, the myc-rearranged band was not associated with the JM rearranged band by £coRI (fig. 3) and Hind III diges tion (data not shown). Northern Blot Analysis. In order to confirm that cmyc and bcl-2 genes were transcribed in KHM-2B cells, Northern analysis of K.HM-2B, fresh lympho blasts and HL 60 cells was performed. Fresh and KHM-2B cells contained a 2.3 kb c-myc transcript similar to that seen in HL 60 cells (fig.4a). On the other hand, two 6-kb and 4-kb mRNAs of bcl-2 were found in fresh and KHM-2B cells but not in HL 60 cells (fig. 4b).
Table 1. Surface markers of leukemic cells from patient’s peripheral blood and the KHM-2B line
HLA-DR CD2 (T il) CD3 (T3) CD4 (T4) CD8 (T8) CDIO(CALLA) CD19 (B4) CD20(B1) slg polyvalent slgy slg a sig n sIg K slgX.
Fresh cell, %
Cell line, %
88.9 23.9 25.2 16.6 18.0 72.5 74.3 73.0 NT NT NT
100.0 4.8 1.4 1.4 1.7 32.9 95.6 52.1 92.6 0.5 0.3 93.3 0.2 85.8
1.1
1.1 3.4
NT = Not tested.
Discussion We have studied an acute lymphocytic leukemiaderived cell line, KHM-2B, that carries an 8; 14 and a 14; 18 translocation with breakpoints similar to those described for Burkitt’s lymphoma [2, 19] and follicu lar lymphoma [10, 20]. As a result of the 8; 14 and 14; 18 translocations, the two immunoglobulin heavy chain genes of the KHM-2B cells were directly in volved in rearrangement with oncogenes derived
from chromosome 8, c-myc and chromosome 18, bcl-2. Rearrangements of the c-myc and bcl-2 were detected by Southern blot analysis. Northern blot analysis also indicated the presence of mRNAs of both oncogenes. Follicular lymphoma with t( 14; 18) translocation is of relatively low-grade malignancy, while Burkitt’s lymphoma with t(8; 14) translocation is of relatively
Cell Line
Fresh Cell
HL60
Fig. 4. Northern blot analysis of 20 pi mRNA from fresh lymphoblast and K.I1M-2B cells. mRNA from IIL60 was used as positive con trol for c-myc mRNA. The filter was hybridized with c-myc (a) and bcl-2 (b) probes. Expression of c-myc (2.3 kb) and bcl-2 (6 and 4 kb) mRNAs was demonstrated.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
Cell Line
Fresh Cell
high-grade malignancy. As postulated by Pegoraro et al. [21] and Gauwerky et al. [22], it is likely that the 14; 18 translocation may have first occurred in an acti vated B cell. This translocation may have placed the bcl-2 gene close to a heavy chain enhancer, leading to the activation and constitutive expression of this pu tative oncogene and the slow induction of the clonal expansion of the B cells with the 14; 18 translocation. Among the neoplastic clones of the proliferating B cells, a translocation between chromosome 8 and 14 might have occurred in a neoplastic B cell as a second genetic change. This second translocation may have resulted in the activation of the translocated c-myc oncogene, leading to a high-grade malignancy. This cell line should be useful for studying multistep on cogenesis. To our knowledge, 4 other cases [21, 23-25] with a t(8; 14) and t( 14; 18) translocation have been reported and one cell line, 380, has been established from the patient by Pegoraro et al. [21], They have analyzed the molecular genetics of the breakpoints involved in the t(8; 14) and t( 14; 18) translocations and speculated that the t( 14; 18) translocation occurred as a mistake in VHD h-J h joining, whereas the t(8; 14) translocation oc curred during heavy chain isotype switching. Both translocations may delete both complete immuno globulin genes on chromosome 14. However, KHM2B cells express surface immunoglobulin (n*A.) dif ferent from that of the 380 cell line. This indicates that the translocation of at least one oncogene occurred without deletion of the immunoglobulin gene. A mo lecular genetic study of the breakpoints of both trans locations of KHM-2B is also needed for understand ing this phenomenon. References 1 Manolov G, Manolova Y: Marker band in one chromosome 14 from Burkitt’s lymphomas. Nature 1972;237:33-34. 2 Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM: Human c-myc one gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 1982;79:7824-7827. 3 Neel BG, Jhanwar SC, Chaganti RS, Hayward WS: Two hu man c-onc genes are located on the long arm of chromosome 8. Proc Natl Acad Sci USA 1982;79:7842-7846. 4 Taub R, Kirsch 1, Morton C, Lenoir G, Swan D, Tronick S, Aaronson S, Leder P: Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Birkitt lymphoma and murine plasmacytoma cells. Proc Natl Acad Sci USA 1982;79:7831-7841. 5 Croce CM, Thierfelder W, Erikson J, Nishikura K, Finan J, Le
6
7
8
9
10
11
12
13
14
15 16
17
18
19
20
21
noir GM, Nowell PC: Transcriptional activation of an unrear ranged and untranslocated c-myc oncogene by translocation of a C lambda locus in Burkitt. Proc Natl Acad Sci USA 1983; 80:6922-6926. Erikson J, Nishikura K, ar-Rushdi A, Finan J, Emanuel B, Le noir G, Nowell PC, Croce CM: Translocation of an immuno globulin kappa locus to a region 3' of an unrearranged c-myc oncogene enhances c-myc transcription. Proc Natl Acad Sci USA 1983;80:7581-7585. Nishikura K, ar-Rushdi A, Erikson J, Watt R, Rovera G, Croce CM: Differential expression of the normal and of the translo cated human c-myc oncogenes in B cells. Proc Natl Acad Sci USA 1983;80:4822-4826. Fukuhara S, Rowley JD, Variakojis D, Golomb HM: Chromo some abnormalities in poorly differentiated lymphocytic lym phoma. Cancer Res 1979:39:3119-3128. Bloomfield CD, Arthur DC, Frizzera G, Levine FG, Peterson BA, Gaji-Peczalska KJ: Nonrandom chromosome abnormali ties in lymphoma. Cancer Res 1983;43:2975-2984. Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR, Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkins’ lymphoma. N Engl J Med 1982;307:1231-1236. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM: Cloning of the chromosome breakpoint of neoplastic B cells with the t( 14; 18) chromosome translocation. Science 1984; 226:1097-1099. Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein AL, Korsmeyer SJ: Cloning the chromosomal break point of t( 14; 18) human lymphomas: Clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 1985;41:898-906. Tsujimoto Y, Cossman J, Jaffe E, Croce CM: Involvement of the bcl-2 gene in human follicular lymphoma. Science 1985; 228:1440-1443. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 1960;20:613-616. Paris Conference (1971): Standardization in human cytogenet ics. Birth Defects. New York,The National Foundation, 1972. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98:503-517. Chirgwin J, Przybla H, MacDonald R, Rutter W: Isolation of biologically active ribonucleic acid from sources enriched in ribonucléase. Biochemistry 1979;18:5294-5299. Flanagan JG, Rabbitts TH: The sequence of a human immu noglobulin epsilon heavy chain constant region gene, and evi dence for three non-allelic genes. EM BO J 1982; 1:655-660. Zech L, Haglund U, Nilsson K, Klein G: Characteristic chromo somal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 1976;17:47-56. Yunis JJ, Oken MM, Theologides A, Howe RB, Kaplan ME: Recurrent chromosomal defects are found in most patients with non-Hodgkin’s lymphoma. Cancer Genet Cytogenet 1984;!: 17-28. Pegoraro L, Palumbo A, Erikson J, Falda M, Giovanazzo B, Emanuel BS, Rovera G, Nowell PC, Croce CM: A 14; 18 and an 8; 14 chromosome translocation in a cell line derived from an
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
M a tsu zak i/H a ta/A so u /S u zu sh im a/A k ah o sh i/Y o sh id a/N ag ak u ra/Ish ii/S an ad a/T ak atsu k i
160
ALL Cel! Line with t(8; 14) and t( 14; 18)
25 Gauwerky CE, Hoxie J, Nowell PC, Croce CM: Pre-B-cell leu kemia with a t(8; 14) and a t( 14; 18) translocation is preceded by follicular lymphoma. Oncogene 1988:2:431-435.
Received: January 2, 1990 Accepted: May 8, 1990 Dr. H. Matsuzaki The Second Department of Internal Medicine Kumamoto University Medical School Honjo 1-1-1 Kumamoto 860 (Japan)
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
acute B-cell leukemia. Proc Natl Acad Sci USA 1984;81 : 7166-7170. 22 Gauwerky CE, Haluska FG, Tsujimoto Y, Nowell PC, Croce CM: Evolution of B-cell malignancy: Pre-B-cell leukemia re sulting from MYC activation in a B-cell neoplasm with a rear ranged BCL 2 gene. Proc Natl Acad Sci USA 1988;85: 8548-8552. 23 Fukuhara S, Rowley J D, Variakojis D, Golomb HM: Chromo some abnormalities in poorly differentiated lymphocytic lym phoma. Cancer Res 1979:39:3119-3128. 24 Mufti GJ, Hamblin TJ, Ocscier DG, Johnson S: Common ALL with pre-B-cell features showing (8:14) and ( 14; 18) chromosome translocations. Blood 1983:62:1142-1146.
161
Acta Haematol 1990;84:156-161
Establishment and Characterization of Acute B-Cell Lymphocytic Leukemia Cell Line Showing (8;14) and (14;18) Chromosome Translocation1 Hiromitsu Matsuzakï', Hiroyuki Hata', Norio A sow', Hitoshi Suzushimaa , Yoshitaka Akalwshï', Minoru Yoshidaa , Shouichi Nagakuraa, Toshinori Ishiih, Isao SanadaK, Kiyoshi Takatsukia a Second Department of Internal Medicine, Kumamoto University Medical School, b Department of Medical Technology, College of Medical Science, Kumamoto University, and c Department of Internal Medicine, Kumamoto National Hospital, Kumamoto, Japan
Key Words. Acute lymphocytic leukemia ■Bcl-2 • Cell line ■Myc • Translocation
Introduction Specific chromosomal translocations are consis tently found in some hematological malignancies. For example, a t(8; 14) (q24;q32) translocation is observed in 70-80% of Burkitt’s lymphomas [1]. Dalla-Favera et al. [2] have shown that the c-myc oncogene, which is located on band q24 of human chromosome 8 [2, 3], translocates to the immunoglobulin heavy chain locus on chromosome 14, which is implicated in Burkitt’s lymphoma with the 8; 14 translocation [4], In a minor proportion of Burkitt’s lymphomas, which exhibit a variant t(2;8) or t(8;22) translocation, the c-myc onco gene is placed in proximity to the k or X light chain constant (CKor C\) regions, respectively [5, 6], The consequence of this chromosome rearrangement is a
1 Supported in part by a Grant-in-Aid for Cancer Research (61-2) from the Ministry of Health and Welfare, and a grant (63770903) from the Ministry of Education, Science and Culture of Japan.
deregulation of the involved c-myc gene, so that it is transcribed constitutively at an elevated level [5-7], and the pathogenesis of this high-grade B-cell malig nancy. Another chromosomal translocation, t( 14; 18) (q32;q21), involving the immunoglobulin heavy chain locus on chromosome 14q32, is found in follicular lymphoma [8-10], Tsujimoto et al. [11] postulated a putative protooncogene, bcl-2, which is normally lo cated on band q21 of chromosome 18 and may be moved into close proximity to a rearranged heavy chain locus in band 14q32. By using a probe for the joining (JH) segment of the immunoglobulin heavy chain locus, they and others have been able to identify a bcl-2 [11, 12]. They also reported that bcl-2 has been found to be rearranged in more than 80% of follicular lymphomas [13]. In this report, we have characterized a cell line, KHM-2B, which was derived from a patient with acute lymphoblastic leukemia, who carries both the 14; 18 and the 8; 14 translocations.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
Abstract. Cell line KHM-2B expressing two oncogene products, c-myc and bcl-2, was established from a pa tient with actute lymphocytic leukemia with an 8; 14 and 14; 18 chromosome translocation. Surface marker studies of the cell line showed that the cells were positive for HLA-DR, CALLA (CD10), B1 (CD20) and B4 (CD19), but negative forTl 1 (CD2). The fresh cells from peripheral blood of the patient had no surface immu noglobulins, whereas KHM-2B cells were positive for p-^type surface immunoglobulin. A cytogenetic analy sis of the cell line revealed two translocations, t (8; 14) (q24;q32) and t( 14; 18)(q32;q21). Rearrangement of the c-myc and bcl-2 genes was detected by Southern blot analysis of the KHM-2B DNA. Northern blot analysis revealed production of c-myc and bcl-2 mRNAs. These results indicated that two oncogenes were activated by two translocations to immunoglobulin genes.
157
ALL Cell Line with t(8; 14) and t( 14; 18)
Materials and Methods
Cell Culture Heparinized peripheral blood from the patient was layered on Ficoll-Conray (specific gravity 1.078) and was centrifuged at 400 g for 30 min. The interphase cells were collected and seeded into cul ture plates at approximately 106 cells/ml after washing with com plete medium. In primary culture and the early passages, RPMI 1640 medium containing 20% fetal calf serum was used, but when the cells began to grow steadily, the medium was changed to RPMI 1640 containing 10% fetal calf serum. The cells were maintained throughout under 5%CO; in humidified air at 37 °C. Immunological Marker Studies Measurement of the following surface markers was carried out: HLA-DR, CD2 (T il), CD3 (T3), CD4 (T4), CD8 (T8), CD10 (CALLA), CD19 (B4), CD20 (Bl) (Becton Dickinson, Mountain View, Calif.), and surface immunoglobulins (slg). Cytogenetics Cytogenetic studies of KHM-2B cells were performed using conventional trypsin-Giemsa chromosome banding techniques [14]. Karyotypes were arranged according to the criteria of the Paris Conference [15]. Southern Blot Analysis DNA was analyzed as described previously [16]. Briefly, 10 pg of high molecular weight DNA was digested with appropriate re striction enzymes, electrophoresed in 0.7% agarose gel and trans ferred to nitrocellulose filters. After being prehybridized at 42°C in plastic sealed bags containing 5 x standard saline citrate (SSC), 5 x Denhardt’s solution, 50% formamide, 20 mAf sodium phosphate buffer and heat-denatured salmon sperm DNA (200 pg/ml), filters were hybridized with a 32P-labeled c-DNA probe at 42°C, and washed 3 times at room temperature with 2xSSC/0.1% sodium dodecyl sulfate and then washed at 56“C with 0.1 x SSC/0.1% so dium dodecyl sulfate. Finally, filters were exposed to X-ray film with intensifying screens overnight at -70°C.
Fig. 1. Cytologic appearance of KHM-2B cells. KHM-2B cells had nuclei containing nucleoli and slightly basophilic cytoplasm with fine vacuoles (Wright-Giemsa stain).
Northern Blot Analysis Total cellular RNA was extracted using the guanidine thiocyanate/cesium chloride method as described elsewhere [17], Ten mi crograms of total RNA was denatured by heating at 60°C for 20 min in 50% (vol/vol) formamide and electrophoresed in 1% aga rose gel containing 2.2 M formaldehyde. After transferring the RNA to nitrocellulose filters, the filters were baked for 2 h at 80°C followed by hybridization, washing and autoradiography as in Southern blotting. DNA Probes The probes used in this study were a 2.5-kilobase (kb) £coRIBgl II fragment containing the Ig heavy chain joining region gene (provided by Dr. T. H. Rabbitts, Cambridge, UK) [18], a 1.8-kb C/al-EcoRI fragment containing the third exon of the human cmyc gene (purchased from ONCOR, Inc., Gaitherburg, Md.), and a 2.8-kb £coRI-Hifu/III fragment containing the bcl-2 gene (pur chased from ONCOR, Inc.).
Results Morphology o f Cultured Cells. The blast cells were collected and cultured from the patient’s peripheral blood before chemotherapy. At early passages, KHM-2B floated in the culture medium as single cells. At a later passage, some of them formed soft
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
Case Report Cell line KHM-2B was derived from the peripheral blood of a 62-year-old man, who was admitted to our hospital in June 1988 for fever, left abdominal pain, diarrhea and anorexia. Physical exami nation revealed only moderate hepatosplenomegaly but no lymph node swelling. Hematologic tests showed an increased leukocyte count (13,500/mm5) with 59% blast cells, a hemoglobin concentra tion of 7.4 g/dl, and a platelet count of 51,000/mm3. The nucleated cell count of bone marrow was 90,000/mm5 including 96.4% blast cells. Using Wright-Giemsa staining, blast cells showed a high nucleo-cytoplasmic ratio, with fine chromatin and several vacuoles in cytoplasm. Blast cells were negative for peroxidase and positive for periodic acid-Schiff stain. On the basis of blast cell morphology, the patient was suspected to have acute lymphoblastic leukemia type L3 (FAB classification). Clinical improvement was achieved transiently by intensive chemotherapy with daunomycin, vincris tine, L-asparaginase and prednisolone. However, increase of drugresistant blast cells and abdominal mass rapidly developed, and the patient died in September 1988. Autopsy revealed acute lympho cytic leukemia, with invasion into widespread lymph nodes, liver, spleen, intestine and peritoneum.
158
M a tsu zak i/H a ta/A so u /S u zu sh im a/A k ah o sh i/Y o sh id a/N ag ak u ra/Ish ii/S an ad a/T ak atsu k i
il
i f
i
2
3
l i 6
I I 7
H 8
» « 13
M I4
n
•
• 19
1
I f 4
1 f 9
• « 16
•
) 5
Il
U I2
11 17
1 * 18
) « XY
22
3
4
1 2
3
Fig. 2. Karyotype of the KHM-2B cells: 46, XY, t(8; 14) (q24;q32), t( 14; 18)(q32;q21), -21, + der(21)t(21; ?)(p 12; ?). As a result of the two reciprocal chromosomal translocations, the KHM-2B cells have two abnormal chro mosomes 14 (14q+), one rearranged chromo some 8 (8q-), and one abnormal chromosome 18(18q—).
4
kb '
»»
:
6.6-
1
l
9.4-
I I
1
2 3.1-
«
4.4-
bel 2
JH
clusters that continued to float. In Wright-Giemsastained smears, KHM-2B cells were morphologically similar to fresh cells, having nuclei containing nucle oli and slightly basophilic cytoplasm with vacuoles (fig- !)• Surface Markers. Fresh cells and KHM-2B cells were positive for HLA-DR, CD10 (CALLA), CD19 (B4), and CD20 (Bl). KHM-2B cells expressed p-A. type slg which was not detected in the freshly isolated cells (table 1). Karyotypic Analysis. Cytogenetic analysis of the
c-myc m
KHM-2B cells is of particular interest. All the cells of 15 metaphases analyzed were found to have t(8;14)(q24;q32) and t( 14; 18)(32;q21) (fig. 2). An addi tional chromosomal aberration such as -21, + der(21)t(21;?)(pl2;?) in figure 2, which was not clonal, was also detected. Southern Blot Analysis. Analysis of gene rearrange ment helped to determine the clonality of the cell line. Fresh lymphoblast cells from the patient and K.HM-2B cells showed the same rearranged bands of immunoglobulin, bcl-2 and c-myc genes (fig. 3). Both
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
2.3-
Fig. 3. Southern blot analysis of DNA from CEM (1), fresh lymphoblast cells (2), KHM-2B cells (3), and patient’s peripheral blood lymphocytes during complete remis sion (4) after digestion with EcoRI. The ni trocellulose filter was hybridized with bcl-2, J„, and c-myc probes. The arrows indicate the rearranged bands.
ALL Cell Line with t(8;14) and t( 14; 18)
159
samples, digested with £coRI, revealed rearranged JH fragments 6.2 and 10.2 kb in size. The size of the bcl-2 rearranged fragment was the same as the 6.2-kb J h rearranged band, which was also confirmed by Hind III digestion (data not shown). On the other hand, the myc-rearranged band was not associated with the JM rearranged band by £coRI (fig. 3) and Hind III diges tion (data not shown). Northern Blot Analysis. In order to confirm that cmyc and bcl-2 genes were transcribed in KHM-2B cells, Northern analysis of K.HM-2B, fresh lympho blasts and HL 60 cells was performed. Fresh and KHM-2B cells contained a 2.3 kb c-myc transcript similar to that seen in HL 60 cells (fig.4a). On the other hand, two 6-kb and 4-kb mRNAs of bcl-2 were found in fresh and KHM-2B cells but not in HL 60 cells (fig. 4b).
Table 1. Surface markers of leukemic cells from patient’s peripheral blood and the KHM-2B line
HLA-DR CD2 (T il) CD3 (T3) CD4 (T4) CD8 (T8) CDIO(CALLA) CD19 (B4) CD20(B1) slg polyvalent slgy slg a sig n sIg K slgX.
Fresh cell, %
Cell line, %
88.9 23.9 25.2 16.6 18.0 72.5 74.3 73.0 NT NT NT
100.0 4.8 1.4 1.4 1.7 32.9 95.6 52.1 92.6 0.5 0.3 93.3 0.2 85.8
1.1
1.1 3.4
NT = Not tested.
Discussion We have studied an acute lymphocytic leukemiaderived cell line, KHM-2B, that carries an 8; 14 and a 14; 18 translocation with breakpoints similar to those described for Burkitt’s lymphoma [2, 19] and follicu lar lymphoma [10, 20]. As a result of the 8; 14 and 14; 18 translocations, the two immunoglobulin heavy chain genes of the KHM-2B cells were directly in volved in rearrangement with oncogenes derived
from chromosome 8, c-myc and chromosome 18, bcl-2. Rearrangements of the c-myc and bcl-2 were detected by Southern blot analysis. Northern blot analysis also indicated the presence of mRNAs of both oncogenes. Follicular lymphoma with t( 14; 18) translocation is of relatively low-grade malignancy, while Burkitt’s lymphoma with t(8; 14) translocation is of relatively
Cell Line
Fresh Cell
HL60
Fig. 4. Northern blot analysis of 20 pi mRNA from fresh lymphoblast and K.I1M-2B cells. mRNA from IIL60 was used as positive con trol for c-myc mRNA. The filter was hybridized with c-myc (a) and bcl-2 (b) probes. Expression of c-myc (2.3 kb) and bcl-2 (6 and 4 kb) mRNAs was demonstrated.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
Cell Line
Fresh Cell
high-grade malignancy. As postulated by Pegoraro et al. [21] and Gauwerky et al. [22], it is likely that the 14; 18 translocation may have first occurred in an acti vated B cell. This translocation may have placed the bcl-2 gene close to a heavy chain enhancer, leading to the activation and constitutive expression of this pu tative oncogene and the slow induction of the clonal expansion of the B cells with the 14; 18 translocation. Among the neoplastic clones of the proliferating B cells, a translocation between chromosome 8 and 14 might have occurred in a neoplastic B cell as a second genetic change. This second translocation may have resulted in the activation of the translocated c-myc oncogene, leading to a high-grade malignancy. This cell line should be useful for studying multistep on cogenesis. To our knowledge, 4 other cases [21, 23-25] with a t(8; 14) and t( 14; 18) translocation have been reported and one cell line, 380, has been established from the patient by Pegoraro et al. [21], They have analyzed the molecular genetics of the breakpoints involved in the t(8; 14) and t( 14; 18) translocations and speculated that the t( 14; 18) translocation occurred as a mistake in VHD h-J h joining, whereas the t(8; 14) translocation oc curred during heavy chain isotype switching. Both translocations may delete both complete immuno globulin genes on chromosome 14. However, KHM2B cells express surface immunoglobulin (n*A.) dif ferent from that of the 380 cell line. This indicates that the translocation of at least one oncogene occurred without deletion of the immunoglobulin gene. A mo lecular genetic study of the breakpoints of both trans locations of KHM-2B is also needed for understand ing this phenomenon. References 1 Manolov G, Manolova Y: Marker band in one chromosome 14 from Burkitt’s lymphomas. Nature 1972;237:33-34. 2 Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM: Human c-myc one gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 1982;79:7824-7827. 3 Neel BG, Jhanwar SC, Chaganti RS, Hayward WS: Two hu man c-onc genes are located on the long arm of chromosome 8. Proc Natl Acad Sci USA 1982;79:7842-7846. 4 Taub R, Kirsch 1, Morton C, Lenoir G, Swan D, Tronick S, Aaronson S, Leder P: Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Birkitt lymphoma and murine plasmacytoma cells. Proc Natl Acad Sci USA 1982;79:7831-7841. 5 Croce CM, Thierfelder W, Erikson J, Nishikura K, Finan J, Le
6
7
8
9
10
11
12
13
14
15 16
17
18
19
20
21
noir GM, Nowell PC: Transcriptional activation of an unrear ranged and untranslocated c-myc oncogene by translocation of a C lambda locus in Burkitt. Proc Natl Acad Sci USA 1983; 80:6922-6926. Erikson J, Nishikura K, ar-Rushdi A, Finan J, Emanuel B, Le noir G, Nowell PC, Croce CM: Translocation of an immuno globulin kappa locus to a region 3' of an unrearranged c-myc oncogene enhances c-myc transcription. Proc Natl Acad Sci USA 1983;80:7581-7585. Nishikura K, ar-Rushdi A, Erikson J, Watt R, Rovera G, Croce CM: Differential expression of the normal and of the translo cated human c-myc oncogenes in B cells. Proc Natl Acad Sci USA 1983;80:4822-4826. Fukuhara S, Rowley JD, Variakojis D, Golomb HM: Chromo some abnormalities in poorly differentiated lymphocytic lym phoma. Cancer Res 1979:39:3119-3128. Bloomfield CD, Arthur DC, Frizzera G, Levine FG, Peterson BA, Gaji-Peczalska KJ: Nonrandom chromosome abnormali ties in lymphoma. Cancer Res 1983;43:2975-2984. Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR, Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkins’ lymphoma. N Engl J Med 1982;307:1231-1236. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM: Cloning of the chromosome breakpoint of neoplastic B cells with the t( 14; 18) chromosome translocation. Science 1984; 226:1097-1099. Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein AL, Korsmeyer SJ: Cloning the chromosomal break point of t( 14; 18) human lymphomas: Clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 1985;41:898-906. Tsujimoto Y, Cossman J, Jaffe E, Croce CM: Involvement of the bcl-2 gene in human follicular lymphoma. Science 1985; 228:1440-1443. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 1960;20:613-616. Paris Conference (1971): Standardization in human cytogenet ics. Birth Defects. New York,The National Foundation, 1972. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98:503-517. Chirgwin J, Przybla H, MacDonald R, Rutter W: Isolation of biologically active ribonucleic acid from sources enriched in ribonucléase. Biochemistry 1979;18:5294-5299. Flanagan JG, Rabbitts TH: The sequence of a human immu noglobulin epsilon heavy chain constant region gene, and evi dence for three non-allelic genes. EM BO J 1982; 1:655-660. Zech L, Haglund U, Nilsson K, Klein G: Characteristic chromo somal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 1976;17:47-56. Yunis JJ, Oken MM, Theologides A, Howe RB, Kaplan ME: Recurrent chromosomal defects are found in most patients with non-Hodgkin’s lymphoma. Cancer Genet Cytogenet 1984;!: 17-28. Pegoraro L, Palumbo A, Erikson J, Falda M, Giovanazzo B, Emanuel BS, Rovera G, Nowell PC, Croce CM: A 14; 18 and an 8; 14 chromosome translocation in a cell line derived from an
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
M a tsu zak i/H a ta/A so u /S u zu sh im a/A k ah o sh i/Y o sh id a/N ag ak u ra/Ish ii/S an ad a/T ak atsu k i
160
ALL Cel! Line with t(8; 14) and t( 14; 18)
25 Gauwerky CE, Hoxie J, Nowell PC, Croce CM: Pre-B-cell leu kemia with a t(8; 14) and a t( 14; 18) translocation is preceded by follicular lymphoma. Oncogene 1988:2:431-435.
Received: January 2, 1990 Accepted: May 8, 1990 Dr. H. Matsuzaki The Second Department of Internal Medicine Kumamoto University Medical School Honjo 1-1-1 Kumamoto 860 (Japan)
Downloaded by: Lund University Libraries 130.235.66.10 - 1/8/2019 2:29:17 PM
acute B-cell leukemia. Proc Natl Acad Sci USA 1984;81 : 7166-7170. 22 Gauwerky CE, Haluska FG, Tsujimoto Y, Nowell PC, Croce CM: Evolution of B-cell malignancy: Pre-B-cell leukemia re sulting from MYC activation in a B-cell neoplasm with a rear ranged BCL 2 gene. Proc Natl Acad Sci USA 1988;85: 8548-8552. 23 Fukuhara S, Rowley J D, Variakojis D, Golomb HM: Chromo some abnormalities in poorly differentiated lymphocytic lym phoma. Cancer Res 1979:39:3119-3128. 24 Mufti GJ, Hamblin TJ, Ocscier DG, Johnson S: Common ALL with pre-B-cell features showing (8:14) and ( 14; 18) chromosome translocations. Blood 1983:62:1142-1146.
161
