Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors

Leukemia Research 30 (2006) 1385–1390

Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors Shuji Tohda a,∗,1 , Taku Sato a , Hanae Kogoshi a , Lu Fu a , Seiji Sakano b , Nobuo Nara a a

Department of Laboratory Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-Ku, Tokyo 113-8519, Japan b Corporate R&D and Central Research Laboratory, Asahi Kasei Corporation, 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan Received 1 November 2005; received in revised form 6 April 2006; accepted 5 May 2006 Available online 14 June 2006

Abstract A novel lymphoma cell line, designated TMD8 was established from cells of a patient with diffuse large B-cell lymphoma. TMD8 cells expressed HES1 mRNA, suggesting constitutive activation of Notch signaling. TMD8 cells expressed normal-sized Notch1 protein, and showed no mutations in the NOTCH1 gene. Cell growth was suppressed by gamma-secretase inhibitors (GSI). It was reported that GSI suppressed growth of T-cell acute lymphoblastic leukemia (T-ALL) cell lines, which frequently had NOTCH1 mutations. In addition to T-ALL, TMD8 is another unique cell line sensitive to GSI, and is useful to study effects of GSI in molecular targeting therapy. © 2006 Elsevier Ltd. All rights reserved. Keywords: Lymphoma; Cell line; Notch; Gamma-secretase inhibitor; Hes1; Jagged

1. Introduction Abnormalities in Notch signaling are involved in various malignancies [1]. Recently, it was reported that more than half of human T cell acute lymphoblastic leukemia (T-ALL) cases had activating mutations in the NOTCH1 gene [2]. Studies also reported that gamma-secretase inhibitors (GSI), which block Notch activation by inhibiting cleavage of the intracellular domain of the Notch protein, induced cell-cycle arrest of T-ALL cell lines. Based on these findings, clinical trials of GSI for refractory T-ALL have begun in the United States. We previously investigated the roles of Notch in abnormal proliferation of leukemia and lymphoma cells [3–7]. We recently established a lymphoma cell line, designated TMD8 from bone marrow cells of a patient suffering from diffuse large B-cell lymphoma. We found that growth of TMD8 cells, which did not show any mutation in the NOTCH1 gene, was suppressed by GSI. Although we have not elucidated the pre∗

Corresponding author. Tel.: +81 3 5803 5334; fax: +81 3 3818 1424. E-mail address: [email protected] (S. Tohda). 1 TMD8 cells can be obtained from our laboratory, on written request to S. Tohda. 0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.05.003

cise mechanisms of suppressive effects of GSI on TMD8 cells, we believe that TMD8 cells are useful for studying effects of GSI on cells, other than T-ALL. Experimental results will shed further information on therapeutic use of GSI for leukemia and lymphomas without NOTCH1 mutations. This study presents characteristics of TMD8 cells.

2. Case history A 62-year-old male was admitted due to high fever and skin rash. Physical findings showed skin plaques, crackles in his right chest, splenomegaly, but no lymphadenopathy. Blood tests revealed a hemoglobin concentration of 7.4 g dl−1 , a platelet count of 22 × 109 l−1 , a white blood cell count of 6.2 × 109 l−1 , an LDH value of 4531 IU l−1 , and a CRP value of 19.1 mg dl−1 . His bone marrow was hypercellular, with 84% lymphoma cells (Fig. 1). Lymphoma cells had a basophilic cytoplasm with abundant vacuoles. Surface antigens are shown in Table 1. Lymphoma cells were also infiltrated in his skin biopsy specimen. At first, the patient was diagnosed as Burkitt lymphoma/leukemia according to morphological findings. The karyotype was 48, XY, add(1)(p3?),

1386

S. Tohda et al. / Leukemia Research 30 (2006) 1385–1390

Fig. 1. Morphology of original lymphoma cells in bone marrow smear (left), and TMD8 cells (right) stained with Wright-Giemsa. Table 1 Immunophenotype of original lymphoma cells in bone marrow aspirate and TMD8 cells Markers

CD3 CD4 CD5 CD7 CD8 CD10 CD13 CD14 CD19 CD20 CD34 CD56 HLA-DR s-IgM s-IgD s-IgG s-␬ s-␭

Positive cells (%) Original cells

TMD8

1 2 70 2 2 1 61 13 67 83 1 1 86 52 0 1 51 1

0 0 48 1 0 4 1 0 91 100 0 1 99 99 1 0 100 1

add(1)(q42), add(6)(p2?), del(6)(q?), +9, i(9)(p10)×2, 15p, +18, −19, +mar. Before the start of chemotherapy, he died of respiratory failure due to pneumonia and lymphoma on the sixth hospital day. Later, diagnosis was altered to diffuse large B-cell lymphoma by the WHO classification [8], because Southern blot analysis showed that the C-MYC gene was not rearranged (data not shown).

3. Materials and methods 3.1. Cell lines Mononuclear cells from the patient’s bone marrow sample were separated by Ficoll-Hypaque density gradient with

informed consent. Cells were cultured in flasks containing ␣-minimal essential medium (␣-MEM; GIBCO, USA) with 10% fetal calf serum (FCS, GIBCO). Cells proliferated immediately, and continuously without addition of growth factors. After 6-month subculture, the established cell line was designated TMD8 in 1999, and used in the following experiments. TMD8 cells have been continuously growing for more than 6 years now. The myeloid leukemia cell lineS TMD7 [4] and THP-1 (supplied from Japanese Cancer Research Resources Bank), which was proliferatively, or suppressively responsive to Notch ligands, respectively [6,7], were used as a control. The T cell acute lymphoblastic leukemia cell line ALL-SIL (gifted from Drs. Harashima and Orita, Fujisaki Cell Center, Okayama, Japan), which was suppressively responsive to GSI [2] and the EBV-positive Burkitt lymphoma cell line Raji (supplied from Japanese Cancer Research Resources Bank) were also used. 3.2. PCR, RT-PCR, and PCR-SSCP For authentication of the established cell line, we examined variable numbers of tandem repeats (VNTR) of the D17S5 locus using polymerase chain reaction (PCR). DNA was extracted from TMD8 cells and the original lymphoma cells. PCR was performed as reported by Dirks et al. [9]. PCR products were electrophoresed on an agarose gel, and stained with ethidium bromide. Detection of EBV DNA in cells was also performed by PCR as reported by Carbone et al. [10]. To examine expressions of NOTCH1–4, JAGGED1, 2, Delta-like (DLL)1, 3, 4, HES1, and EBNA2 mRNAs, reverse transcription (RT)-PCR was performed as reported by us and others [3,5,11–14]. Total RNA was extracted using a RNA isolation kit (Roche Diagnostics, Germany). Firststrand cDNA was synthesized with a First-strand cDNA synthesis kit (Amersham Biosciences, UK). The PCR

S. Tohda et al. / Leukemia Research 30 (2006) 1385–1390

products were electrophoresed and stained with ethidium bromide. To examine mutations of the NOTCH1 gene, nested PCRsingle-strand conformation polymorphism (SSCP) was performed for three regions in a heterodimerization domain, one region in a transcriptional activation domain, and two regions in a PEST domain of the NOTCH1 gene. Primers were synthesized as reported by Weng et al. [2]. PCR products were denatured, electrophoresed through natural polyacrylamide gels [15], and visualized using a silver staining kit (BioRad, USA). 3.3. Proliferation assay Effects of Notch ligands on cell growth were examined by counting cell numbers in culture. Briefly, 1 × 104 cells were cultured in 0.1 ml ␣-MEM with 10% FCS, in 96-well culture plates coated with recombinant Jagged1, Dll1, Dll4, or human IgG1 Fc as control. After 3 days, cells were harvested and cell numbers were counted by the trypan blue method. Recombinant ligands were kimeric proteins composed of the ligand and human IgG1. Ligand preparation and methods of coating on culture plates have been described previously [4,16,17]. Effects of GSI on cell growth were examined in the same way. Three kinds of GSI (Calbiochem, La Jolla, USA), i.e., GSI-I (Z-LLNle-CHO), GSI-IX (DAPT), and GSI-XII (Z-ILCHO), were dissolved in dimethyl sulphoxide (DMSO). Cells were cultured in 96-well plates in the presence of increasing concentrations of GSI. After 2 days, cell numbers were counted.

1387

3.4. Immunoblotting To examine the effect of GSI on the expression and cleavage of Notch1 protein, we performed immunoblotting using an anti-Notch1 antibody (Ab) against the carboxy terminus (Santa Cruz Biotechnology, USA), an anti-cleaved Notch1 (Val1744) Ab (Cell Signaling Technology, USA) to selectively detect a Notch1 intracellular domain (NICD), an active form of Notch1. Before or after cultured with 7.5 ␮M GSIXII for 18 h, cells were harvested and lysed. The lysates from 106 viable cells per lane were subjected to SDS-PAGE and immunoblotted with the antibodies as reported previously [3]. ALL-SIL cells were used as a control for GSI-sensitive cells. 3.5. Quantitative RT-PCR To examine the change in HES1 mRNA expression in cells exposed to Notch ligands or GSI, real-time RT-PCR was performed. Total RNA was extracted from cells cultured with the ligands or human IgG1 Fc, and cells before or after exposure to GSI-I (5 ␮M in 50% acetic acid) for 18 h. We used acetic acid as vehicle for GSI since we had found that DMSO itself affected HES1 mRNA expression. First-strand cDNA was synthesized as described above. Quantitative PCR was performed using a LightCycler FastStart DNA Master SYBR Green I kit, LightCycler primer sets for HES1 and β-ACTIN, and LightCycler ST300 (Roche Diagnostics). HES1 mRNA expression was normalized by β-ACTIN mRNA level measured concurrently.

Fig. 2. Karyotype of TMD8 cells.

1388

S. Tohda et al. / Leukemia Research 30 (2006) 1385–1390

Fig. 3. PCR analysis for variable numbers of tandem repeats in the D17S5 locus (upper panel), and detection of EBV DNA in cells (lower panel). PCR products were electrophoresed, and stained with ethidium bromide. Molecular size markers are shown on the left.

4. Results 4.1. Characterization of TMD8 cells TMD8 cells proliferated singly, or formed small clumps in ␣-MEM with 10% FCS without addition of growth factors. Doubling time was about 30 h. Cells in cytospin preparations (Fig. 1) were of irregular shape, and had a basophilic cytoplasm. Some cells had vacuoles in the cytoplasm. Cell nuclei had one or three nucleoli. Immunophenotype of TMD8 cells was similar to that of original cells, except for absence of CD13 (Table 1). Cell karyotype was 49, XY, add(1)(p3?), add(1)(q42), add(6)(p2?), del(6)(q?), +9, i(9)(p10)×3, 15p, +18, −19, +mar (Fig. 2), which was also similar to that of

Fig. 4. RT-PCR analysis for expression of Notch-related genes and EBNA2 in TMD8 cells. PCR products were electrophoresed, and stained with ethidium bromide.

the original cells. Patterns of VNTR of the D17S5 locus authenticated that TMD8 cells originated from the patient’s cells (Fig. 3, upper panel). EBV DNA was not detected in TMD8 cells (Fig. 3, lower panel). Mycoplasma DNA was not detected (data not shown). 4.2. Notch and the related gene expression in TMD8 cells RT-PCR (Fig. 4) showed that TMD8 cells expressed NOTCH1, NOTCH2, JAGGED2, DLL4 mRNAs. They also expressed HES1 mRNA, suggesting constitutive Notch activation. PCR-SSCP analysis did not detect any mutation in the examined regions of the NOTCH1 gene (data not shown).

Fig. 5. Dose–survival curves for TMD8 cells exposed to gamma-secretase inhibitors (GSI) for 2 days. Data are shown as percent of control. Raji cells were used for comparison.

S. Tohda et al. / Leukemia Research 30 (2006) 1385–1390

4.3. Effects of Notch ligands and GSI on growth of TMD8 cells Stimulations with recombinant Jagged1, Dll1, and Dll4 proteins did not significantly affect growth of TMD8 cells in culture, while they affected the growth of TMD7 and THP-1 cells at the identical concentrations (data not shown). Dose–survival curves for cells exposed to GSI-I and GSIXII are shown in Fig. 5. Survival is shown as percent of control. GSI-I and GSI-XII suppressed growth of TMD8 cells in a dose-dependent manner. DMSO used as vehicle did not affect growth at a concentration corresponding to 10 ␮M GSI (data not shown). Some of the cells cultured with GSI showed nuclear condensation and apoptotic bodies in cytospin preparations, suggesting that GSI induced apoptosis (data not shown). GSI-IX also suppressed the growth dosedependently. The potency was mild (72% decrease at 10 ␮M) compared with GSI-I and GSI-XII.

1389

4.4. Notch protein in cells exposed to GSI Immunoblot analysis (Fig. 6) with anti-Notch1 Ab showed a full-length band (more than 250 kDa) and a transmembrane fragment (120 kDa) in TMD8 cells. Immunoblot with anticleaved Notch1 Ab showed a faint NICD band (100 kDa). GSI treatment did not evidently affect the NICD band. In ALLSIL cells, of which NICD band was much stronger than that of TMD8 cells, GSI obviously decreased the band. 4.5. HES1 mRNA in cells exposed to Notch ligands and GSI To further examine the effect of the ligands and GSI on Notch signaling, HES1 mRNA in TMD8 cells exposed to them was quantified. HES1/β-ACTIN mRNA ratio of control cells was 1.8 × 10−5 . The ratio of cells treated with Jagged1, Dll1, and Dll4 was 2.0 × 10−5 , 2.8 × 10−5 , and 2.6 × 10−5 , respectively. The expression was not significantly increased by any ligand examined. In TMD8 cells before exposure to GSI, HES1/β-ACTIN mRNA ratio was 1.7 × 10−5 . GSI treatment decreased the ratio to 7.0 × 10−6 .

5. Discussion

Fig. 6. Expression of Notch1 proteins in TMD8 cells exposed to GSI. Before or after cultured with GSI-XII for 18 h, cells were harvested and lysed. The lysates (106 viable cells/lane) were subjected to SDS-PAGE, and immunoblotted with anti-Notch1 antibody (Ab), anti-cleaved Notch1 Ab, and anti-␤-actin Ab. ALL-SIL cells were used for comparison. FL, fulllength; NTM, Notch transmembrane fragment; NICD, Notch intracellular domain.

We established the unique new cell line TMD8. The cells expressed HES1 mRNA without ligand stimulation, which suggested that Notch signaling was constitutively active. The growth was suppressed by GSI, a blocker of Notch activation. As for mechanisms of constitutive activation, NOTCH1 mutations, which were frequently observed in T-ALL [2,18], were not recognized. EBNA2, which binds to RBP-J␬ and activates Notch signaling by-passing Notch protein [11,19], was not expressed. We previously reported that some leukemia cells expressed Jagged1 protein, which might cause cell-autonomous Notch activation [3]. Because TMD8 cells expressed JAGGED2 and DLL4 mRNAs, a possible mechanism is the cell-autonomous activation by the ligands expressed on the cells. Exogenous stimulation with recombinant ligands did not significantly promote Notch activation and cell growth. We could not clarify the reason. We surmise that cell-autonomous Notch activation acts on the survival but that Notch activation by exogenous stimulation is irrelevant to the growth. The growth of TMD8 cells was suppressed by GSI-I and GSI-XII at relatively low concentrations, i.e., 5 and 7.5 ␮M, respectively. As a reference, it was reported that the growth of SLK cells, a Kaposi’s sarcoma cell line, was suppressed by 10 ␮M GSI-I [20]. Growth of Ras-transformed fibroblasts was suppressed by 25 ␮M GSI-XII [21]. Some T-ALL cell lines were reported to be sensitive to another GSI, compound E [2]. Our immunoblot analysis showed that treatment with GSI evidently decreased the NICD in ALL-SIL cells. In TMD8 cells, the change in the NICD band was not obviously recognized. Instead, real-time RT-PCR analysis showed that

1390

S. Tohda et al. / Leukemia Research 30 (2006) 1385–1390

GSI treatment decreased HES1 mRNA expression, which meant the suppression of Notch activity. A possibility that GSI inhibited Notch2 activation remains. However, we could not elucidate it because we did not have the methods to assess Notch2 activation. We cannot rule out the possibility that GSI affected some proteins other than Notch in TMD8 cells as there are several gamma-secretase substrates related to cell growth [22]. Further investigation is needed to clarify the mechanism. We are now examining effects of GSI on growth of B-lymphoid and myeloid leukemia cells. We have found that some cell lines without NOTCH1 mutations are GSIsensitive. We hope that our investigations may help to identify a novel molecular target for the treatment of chemotherapyresistant leukemia and lymphomas.

[7]

[8]

[9]

[10]

[11]

Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science. We thank Drs. M. Matsuuchi and Y. Kida for providing clinical data. Contributions. S. Tohda contributed to the concept and design, provided study materials/patients, collected and assembled the data, interpreted and analyzed the data, provided drafting of the article, obtained a funding source, and gave final approval. T. Sato collected and assembled the data. H. Kogoshi collected and assembled the data. L. Fu collected and assembled the data. S. Sakano provided study materials. N. Nara obtained a funding source and gave final approval.

[12]

[13]

[14]

[15]

[16]

References [1] Nickoloff BJ, Osborne BA, Miele L. Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene 2003;22:6598–608. [2] Weng AP, Ferrando AA, Lee W, Morris IV JP, Silverman LB, SanchezIrizarry C, et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 2004;306:269–71. [3] Tohda S, Nara N. Expression of Notch1 and Jagged1 proteins in acute myeloid leukemia cells. Leuk Lymphoma 2001;42:467–72. [4] Tohda S, Sakano S, Ohsawa M, Murakami N, Nara N. A novel cell line derived from de novo acute myeloblastic leukaemia with trilineage myelodysplasia which proliferates in response to a Notch ligand, Delta1 protein. Br J Haematol 2002;117:373–8. [5] Murata-Ohsawa M, Tohda S, Kogoshi H, Sakano S, Nara N. The Notch ligand, Delta-1, alters retinoic acid (RA)-induced neutrophilic differentiation into monocytic and reduces RA-induced apoptosis in NB4 cells. Leuk Res 2005;29:197–203. [6] Tohda S, Murata-Ohsawa M, Sakano S, Nara N. Notch ligands, Delta-1 and Delta-4 suppress the self-renewal capacity and long-term

[17]

[18]

[19]

[20]

[21]

[22]

growth of two myeloblastic leukemia cell lines. Int J Oncol 2003;22: 1073–9. Murata-Ohsawa M, Tohda S, Nara N. Cellular analysis of growth suppression induced by the Notch ligands, Delta-1 and Jagged1 in two myeloid leukemia cell lines. Int J Mol Med 2004;14: 223–6. Diebold J, Jaffe ES, Raphael M, Warnke RA. Burkitt lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. World Health Organization classification of tumors: pathology and genetics of tumors of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2001. p. 181–4. Dirks W, Macleod RAF, Jager K, Milch H, Drexler HG. First searchable database for DNA profiles of human cell lines: sequential use of fingerprint techniques for authentication. Cell Mol Biol 1999;45:841–53. Carbone A, Cilia AM, Gloghini A, Capello D, Todesco M, Quattrone S, et al. Establishment and characterization of EBV-positive and EBV-negative primary effusion lymphoma cell lines harbouring human herpesvirus type-8. Br J Haematol 1998;102:1081–9. Chiaramonte R, Calzavara E, Balordi F, Sabbadini M, Capello D, Gaidano G, et al. Differential regulation of Notch signal transduction in leukemia and lymphoma cells in culture. J Cell Biochem 2003;88:569–77. Zimrin AB, Pepper MS, McMahon GA, Nguyen F, Montesano R, Maciag T. An antisense oligonucleotide to the Notch ligand Jagged enhances fibroblast growth factor-induced angiogenesis in vitro. J Biol Chem 1996;271:32499–502. Masuya M, Katayama N, Hoshino N, Nishikawa H, Sakano S, Araki H, et al. The soluble Notch ligand, Jagged-1, inhibits proliferation of CD34+ macrophage progenitors. Int J Hematol 2002;75:269–76. Zweidler-McKay PA, He Y, Xu L, Rodriguez CG, Karnell FG, Carpenter AC, et al. Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies. Blood 2005;106:3898–906. Oto M, Mitake S, Yuasa Y. Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus. Anal Biochem 1993;213: 19–22. Karanu FN, Murdoch B, Gallacher L, Wu DM, Koremoto M, Sakano S, et al. The Notch ligand Jagged-1 represents a novel growth factor of human hematopoietic stem cells. J Exp Med 2000;192:1365–72. Karanu FN, Murdoch B, Miyabayashi T, Ohno M, Koremoto M, Gallacher L, et al. Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells. Blood 2001;97:1960–7. Lee SY, Kumano K, Masuda S, Hangaishi A, Takita J, Nakazaki K, et al. Mutations of the Notch1 gene in T-cell acute lymphoblastic leukemia: analysis in adults and children. Leukemia 2005;19:1841–3. Hofelmayr H, Strobl LJ, Marschall G, Bornkamm GW, Zimber-Strobl U. Activated Notch1 can transiently substitute for EBNA2 in the maintenance of proliferation of LMP1-expresssing immortalized B cells. J Virol 2001;75:2033–40. Curry CL, Reed LL, Golde TE, Miele L, Nickoloff BJ, Foreman KE. Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi’s sarcoma tumor cells. Oncogene 2005;24: 6333–44. Weijzen S, Rizzo P, Braid M, Vaishnav R, Jonkheer SM, Zolbin A, et al. Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat Med 2002;8:979–86. Kopan R, Ilagan MXG. ␥-Secretase: proteasome of the membrane? Nat Rev Mol Cell Biol 2004;5:499–504.