Cytotoxicity determination without photochemical artifacts

Heuser, Michael; Kopun, Marijana; Rittgen, Werner; Granzow, Christof

doi:10.1016/j.canlet.2004.10.031

Cited by 10 publications

(13 citation statements)

References 25 publications

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“…In vitro cytotoxicity assays were performed as described. 37 Tyrphostin AG 490 (Sigma-Aldrich) was dissolved in ethanol, and added to the culture medium at the specified concentrations as 1/1000th of the final volume.…”

Section: In Vitro Proliferation Assaysmentioning

confidence: 99%

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Heuser

Sly

Argiropoulos

et al. 2009

Blood

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IntroductionSeveral decades ago, variability of leukemias among patients was recognized by morphologic differences and recurrent chromosomal aberrations. In the last decade, a multitude of somatic mutations has been identified in acute myeloid leukemia (AML) patients without cytogenetic aberrations, such as mutations in NPM1, 1 FLT3, 2,3 C/EBP␣, 4 or MLL. 5 Many of these mutations are associated with a distinct prognosis, thus underscoring the heterogeneity of individual leukemias. Heterogeneity has been found not only between but also within individual leukemias. Moreover, in human AML and murine models only a subset of leukemic cells has shown the ability to engraft recipient mice and confer leukemia. 6-9 These findings have provided support for the cancer stem cell (CSC) model, in which tumor growth relies on a subset of cancer cells at the apex of a cellular hierarchy, and in which heterogeneity within the tumor is ascribed to differentiation from the cancer stem cell. 10 Enhanced self-renewal is the hallmark of CSCs and novel treatment approaches aim to specifically target the aberrant self-renewal machinery in CSCs. 11 Whether the heterogeneity among leukemias is represented at the stem cell level has not been systematically investigated. However, understanding the mechanisms underlying aberrant self-renewal in individual leukemias will be of paramount importance to developing leukemia stem cell-specific therapies. To functionally measure leukemia stem cell potential, the operational assay of leukemia-initiating cells (LICs) has been developed. Cells that can initiate the leukemia upon transplantation are denoted LICs, and can be quantified by limiting dilution transplantation. As an approach to modeling leukemias with differing leukemia stem cell properties, we analyzed leukemias engineered by the potent oncogene MN1 alone or in combination with the leukemogenic oncogenes HOXA9 or NUP98-HOXD13. These combinations were chosen based on evidence that these oncogenes may impact distinct pathways in AML patients: we hypothesized that coexpression of MN1 and a HOX gene may recapitulate characteristics of poor risk AML, which presumably is driven by the most aggressive leukemia stem cells, and in which multiple pathways are activated. 12 NPM1-mutated AML, accounting for 50% of patients with cytogenetically normal AML, is characterized by up-regulation of a HOX gene signature and the HOX cofactor MEIS1, whereas NPM1-wild-type AML is characterized by up-regulation of genes such as CD34, MN1, or BAALC that are also expressed in normal hematopoietic stem and progenitor cells. [13][14][15][16] Several HOX genes and MN1 induce myeloid malignancies in mice that represent these NPM1-mutated AML and NPM1-wild-type AML, respectively.The MN1 gene was first identified as a fusion partner of TEL, an ETS transcription factor, in patients with myeloid leukemia or myelodysplastic syndrome containing the translocation t(12; 22)(p13;q11). 17 More recent studies show that MN1 is a potent oncogene producing myeloid malignancy alone...

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Section: In Vitro Proliferation Assaysmentioning

confidence: 99%

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Heuser

Sly

Argiropoulos

et al. 2009

Blood

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“…In vitro cytotoxicity assays and in vitro differentiation assays were performed as described. 1,27 Cells were plated at a density of 4 ϫ 10 4 cells/mL and incubated under light-protective conditions. ATRA (Sigma) was dissolved in DMSO (Sigma), and added to the culture medium at the specified concentrations as 1/1000th of the final volume.…”

Section: In Vitro Assaysmentioning

confidence: 99%

MN1 overexpression induces acute myeloid leukemia in mice and predicts ATRA resistance in patients with AML

Heuser

Argiropoulos

Kuchenbauer

et al. 2007

Blood

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Overexpression of wild-type MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We evaluated whether MN1 plays a functional role in leukemogenesis. We demonstrate using retroviral gene transfer and bone marrow (BM) transplantation that MN1 overexpression rapidly induces lethal AML in mice. Insertional mutagenesis and chromosomal instability were ruled out as secondary aberrations. MN1 increased resistance to all-trans retinoic acid (ATRA)-induced cell-cycle arrest and differentiation by more than 3000-fold in vitro. The differentiation block could be released by fusion of a transcriptional activator (VP16) to MN1 without affecting the ability to immortalize BM cells, suggesting that MN1 blocks differentiation by transcriptional repression. We then evaluated whether MN1 expression levels in patients with AML (excluding M3-AML) correlated with resistance to ATRA treatment in elderly patients uniformly treated within treatment protocol AMLHD98-B. Strikingly, patients with low MN1 expression who received ATRA had a significantly prolonged event-free (P ‫؍‬ .008) and overall (P ‫؍‬ .04) survival compared with patients with either low MN1 expression and no ATRA, or high MN1 expression with or without ATRA. MN1 is a unique oncogene in hematopoiesis that both promotes proliferation/ self-renewal and blocks differentiation, and may become useful as a predictive marker in

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“…The used ex-vivo chemoresponse tests as well as the concomitant KB-cell experiment for verification of the test's reproducibility were carried out under flavin-protecting conditions to avoid flavin-mediated photoreactions [15,16]. As demonstrated by the 14 KB-cell tests, which in general confirmed the predetermined IC 50 values of cisplatin and docetaxel [24,25], the flawless reproducibility has been proven.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the necessary doses which are required to affect the individual tumor's growth are not known. Applying inadequate doses of chemotherapeutics probably also induces further increase of chemo-resistance in HNSCC to the applied agents and possibly additionally also to other potential therapeutics [13][14][15]. There is a high risk to end up with inefficient but nevertheless toxic treatment of HNSCC patients.…”

Section: Introductionmentioning

confidence: 99%

“…However, conventional in-vitro testing was judged to be useless as predictive assay [10,11]. Indeed, more efficient solutions to overcome critical limitations of conventional in-vitro assays by utilizing flavinprotecting conditions [15] to eliminate flavin-mediated photochemical artifacts [16] are available. Advantageously, they are quality-controlled, and allow also for earlier availability (4 days) of data respective to dosages required to achieve the intended response of the individual HNSCC [17,18].…”

Section: Introductionmentioning

confidence: 99%

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Heterogeneity of epithelial and stromal cells of head and neck squamous cell carcinomas in ex vivo chemoresponse

Horn

Wichmann

Mozet

et al. 2009

Cancer Chemother Pharmacol

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Cytotoxicity determination without photochemical artifacts

Cited by 10 publications

References 25 publications

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

MN1 overexpression induces acute myeloid leukemia in mice and predicts ATRA resistance in patients with AML

Heterogeneity of epithelial and stromal cells of head and neck squamous cell carcinomas in ex vivo chemoresponse

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