Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome

Fiedler, Walter; Henke, Rolf‐Peter; Ergün, Süleyman; Schumacher, Udo; Gehling, Ursula M.; Vohwinkel, Gabi; Kilic, Nerbil; Hossfeld, Dieter K.

doi:10.1002/(sici)1097-0142(20000115)88:2<344::aid-cncr14>3.0.co;2-6

Cited by 34 publications

(24 citation statements)

References 26 publications

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“…16 DNA content was assessed by flow cytometry after treatment of ethanol-permeabilized cells with RNAse and staining with propidium iodide. The karyotype was established using standard cytogenetic techniques, synchronization with synchroset (Amplitech, France), heat denaturation and Giemsa staining of R bands.…”

Section: Cell Line Characterization and Dna Preparationsmentioning

confidence: 99%

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study

Lippert¹,

Girodon²,

Hammond³

et al. 2009

Haematologica

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The online version of this article contains a supplementary appendix. BackgroundMany different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results. Design and MethodsJAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. ResultsA first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques -one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean±2SD -with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primerbased specificity could detect 0.2% JAK2V617F. ConclusionsTechniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.Key words: JAK2V617F, quantification, standardization, allele-specific PCR, myeloproliferative diseases, multicenter study. Citation

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Section: Cell Line Characterization and Dna Preparationsmentioning

confidence: 99%

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study

Lippert¹,

Girodon²,

Hammond³

et al. 2009

Haematologica

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“…Cell lines MB-02, 15 MUTZ-8, 16 SKNO-1 17 and UKE-1 18 were generously provided by the original investigators. The cell lines were cultivated according to the protocols described earlier.…”

Section: Human Cell Linesmentioning

confidence: 99%

SOCS2: inhibitor of JAK2V617F-mediated signal transduction

et al. 2008

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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 cytokineindependent MPD clones.

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“…5 According to ARMS and direct sequencing of genomic PCR products, 5/79 cell lines carried the JAK2 V617F mutation (Figure 1a and b): MB-02 derived from a patient with a history of IM and myeloid metaplasia that developed into AML M7; 14 MUTZ-8 established from a patient with AML M4 25 years after the onset of MDS; 18 SET-2 from a patient at the stage JAK2 V617F mutation in cell lines H Quentmeier et al of leukemic transformation of ET; 19 and UKE-1 from a patient with ET, which transformed into acute leukemia. 20 The AML M6-derived cell line HEL was the only JAK2 V617F positive cell line tested lacking a known patient-history of MPD or MDS. 14 However, like other JAK2mu cell lines found in this study, also this cell line showed cytogenetic rearrangements, which are considered hallmarks of AML/MDS: 21 these included losses of long-arm material from both chromosomes 5 or 7 (5qÀ/7qÀ) affecting cell lines HEL, MB-02, MUTZ-8, and SET-2 (Figure 2a-d), while cell line UKE1 displayed monosomy for chromosome 7 (data not shown).…”

Section: Cell Lines With Jak2 V617f Mutationsmentioning

confidence: 99%

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

et al. 2006

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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.

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Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome

Cited by 34 publications

References 26 publications

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study

SOCS2: inhibitor of JAK2V617F-mediated signal transduction

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

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