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Establishment and characterization of a novel MYC/BCL2 "double-hit" diffuse large B cell lymphoma cell line, RC.

Pham LV, Lu G, Tamayo AT, Chen J, Challagundla P, Jorgensen JL, Medeiros LJ, Ford RJ - J Hematol Oncol (2015)

Bottom Line: In addition, RC cells were established in SCID mice as an in vivo model for translational therapeutics studies.Proteomic analysis showed activation of the mTOR signaling pathway in RC cells that can be targeted with an mTOR inhibitor.The data presented confirm the validity of the RC cell line as a representative model of MYC/BCL2 DHL that will be useful for both in vitro and in vivo studies of DHL pathogenesis and therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd. Unit 54, Houston, TX, 77030, USA. lvpham@mdanderson.org.

ABSTRACT

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoid malignancy worldwide. Approximately 5 % of cases of DLBCL are so-called double-hit lymphomas (DHL), defined by a chromosomal translocation or rearrangement involving MYC/8q24.2 in combination with another recurrent breakpoint, usually BCL2/18q21.3. Patients with MYC/BCL2 DHL are resistant to standard front-line therapy, and currently, there is no consensus for a therapeutic strategy to treat these patients. Lack of clinically relevant or validated human experimental DHL models of any type that would improve our understanding of the biologic basis of MYC/BCL2 DHL pathophysiology continues to hamper identification of valid therapeutic targets. We describe a unique MYC/BCL2 DHL cell line with morphologic features of DLBCL that we have established, designated as RC.

Methods: We used tissue culture techniques to establish the RC cell line from primary DLBCL cells. We also utilized molecular and cellular biological techniques including flow cytometry, polymerase chain reaction (PCR), DNA fingerprinting, reverse-phase protein array, conventional cytogenetics, and fluorescence in situ hybridization (FISH) analysis to characterize the RC cell line. NSG-severe combined immunodeficiency (SCID) mice were utilized as a model for xeno-transplantation of RC cells.

Results: RC cells had the following immunophenotype: positive for CD10, CD19, CD20, CD22, CD38, CD43, CD44, and CD79b and negative for CD3, CD4, CD5, CD8, CD11c, CD14, CD30, CD56, and CD200, which was identical to the primary tumor cells. Conventional cytogenetic analysis showed a t(2;8)(p12;q24.2) and t(14;18)(q32;q21.3), corresponding to MYC and BCL2 gene rearrangements, respectively. DNA fingerprinting authenticated the RC cell line to be of the same clone as the primary tumor cells. In addition, RC cells were established in SCID mice as an in vivo model for translational therapeutics studies. Proteomic analysis showed activation of the mTOR signaling pathway in RC cells that can be targeted with an mTOR inhibitor.

Conclusion: The data presented confirm the validity of the RC cell line as a representative model of MYC/BCL2 DHL that will be useful for both in vitro and in vivo studies of DHL pathogenesis and therapeutics.

No MeSH data available.


Related in: MedlinePlus

Morphologic and phenotypic features of RC cells. a Distribution of the size (longest diameter) of RC cells after 16 months of cell culturing. b Representative image of H&E-stained RC cells after 16 months in cell culture. c PCR analysis for EBV type 1 (EBNA1) and type 2 (EBNA2) gene in Mino (negative control), Granta (positive control), and RC cell lines. GAPDH serves as a loading control
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Fig1: Morphologic and phenotypic features of RC cells. a Distribution of the size (longest diameter) of RC cells after 16 months of cell culturing. b Representative image of H&E-stained RC cells after 16 months in cell culture. c PCR analysis for EBV type 1 (EBNA1) and type 2 (EBNA2) gene in Mino (negative control), Granta (positive control), and RC cell lines. GAPDH serves as a loading control

Mentions: Primary cells were obtained from a pleural effusion of a patient diagnosed with diffuse large B cell lymphoma with high-grade features (high mitotic activity and proliferation rate). The primary cells were washed, explanted, and cultured at approximately 5 × 106 cells/mL in RPMI-1640 media, supplemented with 15 % fetal bovine serum (FBS) without any external stimulation. The primary cells remained viable (~90–95 %) even after 4 weeks in cell culture; however, the number of cells remained constant. During the fifth week in culture, cell number began to increase and identifiable mitotic figures began to appear. From this timepoint, the cells doubled in number every 4–5 days. This established lymphoma cell line successfully continued cell proliferation in a single-cell suspension without cellular clump formation, growing in continuous culture for more than 16 months, and aliquot samples could be frozen in medium composed of 90 % FBS and 10 % DMSO. The cell line was designated as the “RC” cell line, optimally maintained at a density between 1 and 2 × 106 cells/mL and could be split 1:2 every 3–4 days. RC cells are medium-to-large, blast-like lymphoid cells, approximately 9–14 μm in largest diameter (Fig. 1a) with moderately abundant strongly basophilic cytoplasm. The nuclei were round to ovoid with coarse chromatin and occasional irregular nuclear contours. The morphologic features of RC cells were stable and did not change during 16 months in culture (Fig. 1b).Fig. 1


Establishment and characterization of a novel MYC/BCL2 "double-hit" diffuse large B cell lymphoma cell line, RC.

Pham LV, Lu G, Tamayo AT, Chen J, Challagundla P, Jorgensen JL, Medeiros LJ, Ford RJ - J Hematol Oncol (2015)

Morphologic and phenotypic features of RC cells. a Distribution of the size (longest diameter) of RC cells after 16 months of cell culturing. b Representative image of H&E-stained RC cells after 16 months in cell culture. c PCR analysis for EBV type 1 (EBNA1) and type 2 (EBNA2) gene in Mino (negative control), Granta (positive control), and RC cell lines. GAPDH serves as a loading control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4627381&req=5

Fig1: Morphologic and phenotypic features of RC cells. a Distribution of the size (longest diameter) of RC cells after 16 months of cell culturing. b Representative image of H&E-stained RC cells after 16 months in cell culture. c PCR analysis for EBV type 1 (EBNA1) and type 2 (EBNA2) gene in Mino (negative control), Granta (positive control), and RC cell lines. GAPDH serves as a loading control
Mentions: Primary cells were obtained from a pleural effusion of a patient diagnosed with diffuse large B cell lymphoma with high-grade features (high mitotic activity and proliferation rate). The primary cells were washed, explanted, and cultured at approximately 5 × 106 cells/mL in RPMI-1640 media, supplemented with 15 % fetal bovine serum (FBS) without any external stimulation. The primary cells remained viable (~90–95 %) even after 4 weeks in cell culture; however, the number of cells remained constant. During the fifth week in culture, cell number began to increase and identifiable mitotic figures began to appear. From this timepoint, the cells doubled in number every 4–5 days. This established lymphoma cell line successfully continued cell proliferation in a single-cell suspension without cellular clump formation, growing in continuous culture for more than 16 months, and aliquot samples could be frozen in medium composed of 90 % FBS and 10 % DMSO. The cell line was designated as the “RC” cell line, optimally maintained at a density between 1 and 2 × 106 cells/mL and could be split 1:2 every 3–4 days. RC cells are medium-to-large, blast-like lymphoid cells, approximately 9–14 μm in largest diameter (Fig. 1a) with moderately abundant strongly basophilic cytoplasm. The nuclei were round to ovoid with coarse chromatin and occasional irregular nuclear contours. The morphologic features of RC cells were stable and did not change during 16 months in culture (Fig. 1b).Fig. 1

Bottom Line: In addition, RC cells were established in SCID mice as an in vivo model for translational therapeutics studies.Proteomic analysis showed activation of the mTOR signaling pathway in RC cells that can be targeted with an mTOR inhibitor.The data presented confirm the validity of the RC cell line as a representative model of MYC/BCL2 DHL that will be useful for both in vitro and in vivo studies of DHL pathogenesis and therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd. Unit 54, Houston, TX, 77030, USA. lvpham@mdanderson.org.

ABSTRACT

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoid malignancy worldwide. Approximately 5 % of cases of DLBCL are so-called double-hit lymphomas (DHL), defined by a chromosomal translocation or rearrangement involving MYC/8q24.2 in combination with another recurrent breakpoint, usually BCL2/18q21.3. Patients with MYC/BCL2 DHL are resistant to standard front-line therapy, and currently, there is no consensus for a therapeutic strategy to treat these patients. Lack of clinically relevant or validated human experimental DHL models of any type that would improve our understanding of the biologic basis of MYC/BCL2 DHL pathophysiology continues to hamper identification of valid therapeutic targets. We describe a unique MYC/BCL2 DHL cell line with morphologic features of DLBCL that we have established, designated as RC.

Methods: We used tissue culture techniques to establish the RC cell line from primary DLBCL cells. We also utilized molecular and cellular biological techniques including flow cytometry, polymerase chain reaction (PCR), DNA fingerprinting, reverse-phase protein array, conventional cytogenetics, and fluorescence in situ hybridization (FISH) analysis to characterize the RC cell line. NSG-severe combined immunodeficiency (SCID) mice were utilized as a model for xeno-transplantation of RC cells.

Results: RC cells had the following immunophenotype: positive for CD10, CD19, CD20, CD22, CD38, CD43, CD44, and CD79b and negative for CD3, CD4, CD5, CD8, CD11c, CD14, CD30, CD56, and CD200, which was identical to the primary tumor cells. Conventional cytogenetic analysis showed a t(2;8)(p12;q24.2) and t(14;18)(q32;q21.3), corresponding to MYC and BCL2 gene rearrangements, respectively. DNA fingerprinting authenticated the RC cell line to be of the same clone as the primary tumor cells. In addition, RC cells were established in SCID mice as an in vivo model for translational therapeutics studies. Proteomic analysis showed activation of the mTOR signaling pathway in RC cells that can be targeted with an mTOR inhibitor.

Conclusion: The data presented confirm the validity of the RC cell line as a representative model of MYC/BCL2 DHL that will be useful for both in vitro and in vivo studies of DHL pathogenesis and therapeutics.

No MeSH data available.


Related in: MedlinePlus