High level EGFR amplification in a newly established glioblastoma cell line 170-MG-BA.

Glioblastoma multiforme is a highly invasive and incurable primary brain tumor. The most frequent genetic alteration therein is amplification of the epidermal growth factor receptor (EGFR) gene, the target of current clinical trials. However, EGFR amplification is poorly represented in glioblastoma cell lines. From the 30 cultures attempted herein, we were able to establish two glioblastoma permanent cell lines. The remaining cultures showed limited life span and underwent senescence between passage numbers (PN) 8 to 15. Our newly established glioblastoma cell lines, designated 170-MG-BA and 538-MG-BA, both originated between PN 3 and 5 when areas of smaller, more rapidly proliferating cells appeared. Both cell lines showed similar rates of growth, moderate morphological differences, cytoskeletal heterogeneity and multiple chromosome rearrangements. Analysis by molecular cytogenetics and comparative genomic hybridization (aCGH) revealed two copies of a stable marker chromosome in 170-MG-BA cells effecting focal amplification at 7q11 of the EGFR locus. Comparative RqPCR analysis confirmed that EGFR was uniquely highly expressed in 170-MG-BA cells. Combined targeted expression analysis and aCGH data excluded the recurrent EGFRvIII activating mutation. In contrast, EGFR expression in 538-MG-BA cells which lacked genomic EGFR amplification was not raised. Immunofluorescent staining showed high EGFR protein expression only in the 170-MG-BA cells. Cytogenetic, genomic and transcriptional analyses then confirmed high-level genomic amplification and transcriptional upregulation of wild type EGFR in 170-MG-BA; the first conventional cell line model for investigating the biology and targeted therapy of this key alteration in glioblastoma. Both cell lines are freely available from the DSMZ cell repository.

U-2932: two clones in one cell line, a tool for the study of clonal evolution.

Genetic heterogeneity is common in tumors, explicable by the development of subclones with distinct genetic and epigenetic alterations. We describe an in vitro model for cancer heterogeneity, comprising the diffuse large B-cell lymphoma cell line U-2932 which expresses two sets of cell surface markers representing twin populations flow-sorted by CD20 vs CD38 expression. U-2932 populations were traced to subclones of the original tumor with clone-specific immunoglobulin IgVH4-39 hypermutation patterns. BCL6 was overexpressed in one subpopulation (R1), MYC in the other (R2), both clones overexpressed BCL2. According to the combined results of immunoglobulin hypermutation and cytogenetic analysis, R1 and R2 derive from a mother clone with genomic BCL2 amplification, which acquired secondary rearrangements leading to the overexpression of BCL6 (R1) or MYC (R2). Some 200 genes were differentially expressed in R1/R2 microarrays including transcriptional targets of the aberrantly expressed oncogenes. Other genes were regulated by epigenetic means as shown by DNA methylation analysis. Ectopic expression of BCL6 in R2 variously modulated new candidate target genes, confirming dual silencing and activating functions. In summary, stable retention of genetically distinct subclones in U-2932 models tumor heterogeneity in vitro permitting functional analysis of oncogenes against a syngenic background.

SOCS2: inhibitor of JAK2V617F-mediated signal transduction.

Janus kinase 2 (JAK2)V617F-activating mutations (JAK2mu) occur in myeloproliferative disorders (MPDs) and myelodysplastic syndromes (MDSs). Cell lines MB-02, MUTZ-8, SET-2 and UKE-1 carry JAK2V617F and derive from patients with MPD/MDS histories. Challenging the consensus that expression of JAK2V617F is the sole precondition for cytokine independence in class I cytokine receptor-positive cells, two of four of the JAK2mu cell lines were growth factor-dependent. These cell lines resembled JAK2wt cells regarding JAK2/STAT5 activation: cytokine deprivation effected dephosphorylation, whereas erythropoetin or granulocyte colony-stimulating factor induced phosphorylation of JAK2 and STAT5. Cytokine independence correlated with low expression and cytokine dependence with high expression of the JAK/STAT pathway inhibitor suppressor of cytokine signaling 2 (SOCS2) suggesting a two-step mechanism for cytokine independence of MPD cells: (i) activation of the oncogene JAK2V617F and (ii) inactivation of the tumor suppressor gene SOCS2. Confirming that SOCS2 operates as a negative JAK2V617F regulator, SOCS2 knockdown induced constitutive STAT5 phosphorylation in JAK2mu cells. CpG island hypermethylation is reported to promote SOCS gene silencing in malignant diseases. Accordingly, in one of two cytokine-independent cell lines and in two of seven MPD patients, we found SOCS2 hypermethylation associated with reduced promoter access to transcription factors. Our results provide solid evidence that SOCS2 epigenetic downregulation might be an important second step in the genesis of cytokine-independent MPD clones.

MEF2C is activated by multiple mechanisms in a subset of T-acute lymphoblastic leukemia cell lines.

In T-cell acute lymphoblastic leukemia (T-ALL) the cardiac homeobox gene NKX2-5 (at 5q35) is variously deregulated by regulatory elements coordinating with BCL11B (at 14q32.2), or the T-cell receptor gene TRD (at 14q11.2), respectively. NKX2-5 is normally expressed in developing spleen and heart, regulating fundamental processes, including differentiation and survival. In this study we investigated whether NKX2-5 expression in T-ALL cell lines reactivates these embryonal pathways contributing to leukemogenesis. Among 18 known targets analyzed, we identified three genes regulated by NKX2-5 in T-ALL cells, including myocyte enhancer factor 2C (MEF2C). Knockdown and overexpression assays confirmed MEF2C activation by NKX2-5 at both the RNA and protein levels. Direct interactions between NKX2-5 and GATA3 as indicated by co-immunoprecipitation data may contribute to MEF2C regulation. In T-ALL cell lines LOUCY and RPMI-8402 MEF2C expression was correlated with a 5q14 deletion, encompassing noncoding proximal gene regions. Fusion constructs with green fluorescent protein permitted subcellular detection of MEF2C protein in nuclear speckles interpretable as repression complexes. MEF2C consistently inhibits expression of NR4A1/NUR77, which regulates apoptosis via BCL2 transformation. Taken together, our data identify distinct mechanisms underlying ectopic MEF2C expression in T-ALL, either as a downstream target of NKX2-5, or via chromosomal aberrations deleting proximal gene regions.

Amplification at 7q22 targets cyclin-dependent kinase 6 in T-cell lymphoma.

Recurrent chromosomal aberrations in hematopoietic tumors target genes involved in pathogenesis. Their identification and functional characterization are therefore important for the establishment of rational therapies. Here, we investigated genomic amplification at 7q22 in the T-cell lymphoma cell line SU-DHL-1 belonging to the subtype of anaplastic large-cell lymphoma (ALCL). Cytogenetic analysis mapped this amplicon to 86-95 Mb. Copy-number determination quantified the amplification level at 5- to 6-fold. Expression analysis of genes located within this region identified cyclin-dependent kinase 6 (CDK6) as a potential amplification target. In comparison with control cell lines, SU-DHL-1 expressed considerably higher levels of CDK6. Functionally, SU-DHL-1 cells exhibited reduced sensitivity to rapamycin treatment, as indicated by cell growth and cell cycle analysis. Rapamycin reportedly inhibits degradation of the CDK inhibitor p27 with concomitant downregulation of cyclin D3, implying a proliferative advantage for CDK6 overexpression. Amplification of the CDK6 locus was analyzed in primary T-cell lymphoma samples and, while detected infrequently in those classified as ALCL (1%), was detected in 23% of peripheral T-cell lymphomas not otherwise specified. Taken together, analysis of the 7q22 amplicon identified CDK6 as an important cell cycle regulator in T-cell lymphomas, representing a novel potential target for rational therapy.

Human T-cell lines with well-defined T-cell receptor gene rearrangements as controls for the BIOMED-2 multiplex polymerase chain reaction tubes.

The BIOMED-2 multiplex polymerase chain reaction (PCR) tubes for analysis of immunoglobulin and T-cell receptor (TCR) gene rearrangements have recently been introduced as a reliable and easy tool for clonality diagnostics in suspected lymphoproliferations. Quality and performance assessment of PCR-based clonality diagnostics is generally performed using human leukemia/lymphoma cell lines as controls. We evaluated the utility of 30 well-defined human T-cell lines for quality performance testing of the BIOMED-2 PCR primers and protocols. The PCR analyses of the TCR loci were backed up by Southern blot analysis. The clonal TCRB, TCRG and TCRD gene rearrangements were analyzed for gene segment usage and for the size and composition of their junctional regions. In 29 out of 30 cell lines, unique clonal TCR gene rearrangements could be easily detected. Besides their usefulness in molecular clonality diagnostics, these cell lines can now be authenticated based on their TCR gene rearrangement profile. This enables their correct use in molecular clonality diagnostics and in other cancer research studies.

JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.

A mutation in the JH2 pseudokinase domain of the Janus kinase 2 gene (JAK2 V617F) has been described in chronic myeloproliferative disorders (MPD). We screened 79 acute myeloid leukemia (AML) cell lines and found five positive for JAK2 V617F (HEL, MB-02, MUTZ-8, SET-2, UKE-1), 4/5 with histories of MPD/MDS. While SET-2 expressed both mutant (mu) and wild-type (wt) JAK2, remaining positives carried homo-/hemizygous JAK2 mutations. Microsatellite analysis confirmed losses of heterozygosity (LOH) affecting the JAK2 region on chromosome 9p in MB-02, MUTZ-8 and UKE-1, but also in HEL, the only JAK2mu cell line lacking any reported MPD/MDS history. All five JAK2mu cell lines displayed cytogenetic hallmarks of MDS, namely losses of 5q or 7q, remarkably in 4/5 cases affecting both chromosomes. Our combined FISH and microsatellite analysis uncovered a novel mechanism to supplement mitotic recombination previously proposed to explain JAK2 LOH, namely chromosome deletion with/without selective JAK2mu amplification. Confirming the importance of the mutated JAK2 protein for growth and prevention of apoptosis, JAK2mu cell lines displayed higher sensitivities to JAK2 inhibition than JAK2wt cell lines. In summary, JAK2 V617F cell lines, derived from patients with history of MPD/MDS, represent novel research tools for elucidating the pathobiology of this JAK2 mutation.

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin.

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

HLXB9 activates IL6 in Hodgkin lymphoma cell lines and is regulated by PI3K signalling involving E2F3.

Multiple cytokines are secreted by Hodgkin lymphoma (HL) cells, notably interleukin-6 (IL6), which is believed to play a significant pathobiological role in this and certain other tumors. Previous work on prostate carcinoma cells has shown that IL6 expression is activated therein by the homeodomain protein GBX2, which we found to be absent in HL cells. Instead, we observed expression of a closely related gene, HLXB9, albeit restricted to HL cells coexpressing IL6. Treatment of HL cell lines with antisense-oligonucleotides directed against HLXB9, forced expression of recombinant HLXB9, and analysis of reporter gene constructs containing IL6 promoter sequences all confirmed the potential of HLXB9 to drive expression of IL6. Chromosomal rearrangements of the HLXB9 locus at 7q36 were not detected in HL cells unlike AML subsets expressing HLXB9. However, inhibition of certain signal transduction pathways revealed that the phosphatidylinositol 3 kinase (PI3K) pathway contributes to HLXB9 expression. AKT/phospho-AKT analysis revealed constitutively active PI3K signalling in HL cell lines. Downstream analysis of PI3K revealed that E2F3 may mediate activation of HLXB9. Taken together, our data show that the PI3K signalling pathway in HL cells is constitutively activated and promotes HLXB9 expression, probably via E2F3, thereby enhancing malignant expression of IL6.

Fusion of NUP214 to ABL1 on amplified episomes in T-cell acute lymphoblastic leukemia.

In T-cell acute lymphoblastic leukemia (T-ALL), transcription factors are known to be deregulated by chromosomal translocations, but mutations in protein tyrosine kinases have only rarely been identified. Here we describe the extrachromosomal (episomal) amplification of ABL1 in 5 of 90 (5.6%) individuals with T-ALL, an aberration that is not detectable by conventional cytogenetics. Molecular analyses delineated the amplicon as a 500-kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (refs. 5,6). We identified a previously undescribed mechanism for activation of tyrosine kinases in cancer: the formation of episomes resulting in a fusion between NUP214 and ABL1. We detected the NUP214-ABL1 transcript in five individuals with the ABL1 amplification, in 5 of 85 (5.8%) additional individuals with T-ALL and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib. The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased HOX expression and deletion of CDKN2A, consistent with a multistep pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of individuals with T-ALL who could benefit from treatment with imatinib.

Malignant hematopoietic cell lines: in vitro models for the study of MLL gene alterations.

Human tumor cell lines are powerful tools for investigating basic and applied aspects of cell biology. Leukemia-lymphoma cell lines have been instrumental in the cytogenetic and molecular analysis of recurring chromosome rearrangements, notably translocations and inversions, thus illuminating the pathogenesis of hematological malignancy. Chromosomal translocations targeting the MLL gene at 11q23 have come to represent a paradigm in acute leukemias. These translocations result in the in-frame joining of the MLL gene with a partner gene to generate unique fusion proteins of putatively novel function. More than 30 partner genes that participate with MLL in the more than 60 known 11q23 translocations have been reported. Cell lines provide territory to both explore the detailed structures of 11q23 translocations and investigate the leukemogenic activities of MLL fusion proteins. We review here the leukemia cell lines that have been described to carry 11q23 translocations and MLL fusion genes. Except for the t(10;11)(p12;q23), each of the following relatively frequent 11q23/MLL translocations is represented by one or more cell lines: 16 cell lines with t(4;11)(q21;q23), two cell lines with t(6;11)(q27;q23), seven cell lines with t(9;11)(p22;q23), and eight cell lines with t(11;19)(q23;p13). For each of three rare translocations, one cell line has been reported: t(5;11)(q15;q23), t(11;16)(q23;p13), and t(X;11)(q13;q23). Of these 36 cell lines with 11q23 translocations, 17 have been made available to us; we confirmed the occurrence of the alterations reported in these cell lines at the chromosomal and/or gene level. A second type of MLL gene alteration is the partial tandem duplication (PTD), which occurs in acute myeloid leukemia (AML). We found four AML cell lines with an MLL PTD; one acute lymphoblastic leukemia-derived cell line was reported to show a partial nontandem duplication. Finally, a third rearrangement involves intrachromosomal amplification of the unrearranged MLL gene leading to multiple copies of the gene and (presumably) increased expression. Three cell lines carrying such MLL amplifications have been described. The availability of these cell lines as model systems provides the opportunity to explore the altered expression or functions of MLL genes and their partners in oncogenesis.

Cloning and genomic organization of the TTL gene on mouse chromosome 2 and human chromosome 2q13.

Tubulin tyrosine ligase (TTL) is a cytosolic enzyme involved in the posttranslational modification of tubulin. In the assembled form microtubules are detyrosinated over time at the C-terminus of alpha-tubulin. After microtubular disassembly TTL restores tyrosine residues back to the detyrosinated tubulin leading to a cycle of detyrosination/tyrosination. Here we report the isolation of the human and mouse TTL cDNA. In comparison with other known TTL sequences, namely bovine, rat and porcine, we found that only porcine TTL deviates in length by having an insertion of two glutamate residues. In mouse and human TTL the genomic coding sequence is composed of seven exons with normal intron/exon boundaries. Using fluorescence in situ hybridization (FISH), we mapped the murine TTL gene to mouse chromosome 2 (MMU2). Human TTL has been located to chromosome 2q13 (HSA2q13). In addition, we found frequently truncated PCR products of hTTL transcripts with aberrant splicing in tumors.