<i>TLX1 (HOX11)</i> Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors

Hawley, Robert G.; Hawley, Teresa S.; Cantor, Alan B.

doi:10.1002/9780470151808.sc01f07s7

Cited by 6 publications

(7 citation statements)

References 42 publications

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“…We and others have reported that enforced expression of TLX1 promotes the immortalization of murine hematopoietic progenitors derived from bone marrow as well as from fetal liver, yolk sac and in vitro differentiated embryonic stem cells from various mouse strains at relatively high frequency (reviewed in Hawley et al, 2008). One caveat of these studies is that gene delivery via MLV-based MSCV retroviral vectors was used to introduce the TLX1 transgene into the target cell populations.…”

Section: Discussionmentioning

confidence: 99%

“…The potential of TLX1 to disrupt normal hematopoietic processes has also been demonstrated in a number of in vitro studies, where retroviral expression of TLX1 promoted the immortalization of murine progenitor cells derived from various hematopoietic sources (Hawley et al, 1994a; Hawley et al, 1997; Keller et al, 1998; Yu et al, 2002; Owens et al, 2003; Yu et al, 2003; reviewed in Hawley et al, 2008). Structure-function analysis of the TLX1 protein showed that an intact homeodomain is necessary for hematopoietic progenitor cell immortalization but regions near the NH 2 and COOH termini of TLX1 are not essential for immortalizing activity (Hawley et al, 1997; Owens et al, 2003).…”

Section: Introductionmentioning

confidence: 93%

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Hematopoietic immortalizing function of the NKL‐subclass homeobox geneTLX1

Zweier-Renn

Hawley

Burkett

et al. 2009

Genes Chromosomes & Cancer

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Translocations resulting in ectopic expression of the TLX1 homeobox gene (previously known as HOX11) are recurrent events in human T-cell acute lymphoblastic leukemia (T-ALL). Transduction of primary murine hematopoietic stem/progenitor cells with retroviral vectors expressing TLX1 readily yields immortalized hematopoietic progenitor cell lines. Understanding the processes involved in TLX1-mediated cellular immortalization should yield insights into the growth and differentiation pathways altered by TLX1 during the development of T-ALL. In recent clinical gene therapy trials, hematopoietic clonal dominance or T-ALL-like diseases have occurred as a direct consequence of insertional activation of the EVI1, PRDM16 or LMO2 proto-oncogenes by the retroviral vectors used to deliver the therapeutic genes. Additionally, the generation of murine hematopoietic progenitor cell lines due to retroviral integrations into Evi1 or Prdm16 has also been recently reported. Here we determined by linker-mediated nested polymerase chain reaction the integration sites in 8 TLX1-immortalized hematopoietic cell lines. Notably, no common integration site was observed among the cell lines. Moreover, no insertions into the Evi1 or Prdm16 genes were identified although insertion near Lmo2 was observed in one instance. However, neither Lmo2 nor any of the other genes examined surrounding the integration sites showed differential vector-influenced expression compared to the cell lines lacking such insertions. While we cannot exclude the possibility that insertional side effects transiently provided a selective growth/survival advantage to the hematopoietic progenitor populations, our results unequivocally rule out insertions into Evi1 and Prdm16 as being integral to the TLX1-initiated immortalization process.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

Hematopoietic immortalizing function of the NKL‐subclass homeobox geneTLX1

Zweier-Renn

Hawley

Burkett

et al. 2009

Genes Chromosomes & Cancer

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“…(i) Labeling of apo-Tf. We labeled Tf by following the procedure described in references 7 [FBS], 100 U/ml penicillin-streptomycin, 2 mM glutamine, 10 ng/ml interleukin-3 [IL-3], 2 ng/ml IL-6, 5 ng/ml IL-11, 50 ng/ml c-kit ligand) for 5 days (25).…”

Section: Methodsmentioning

confidence: 99%

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

Liu

Ciocea

Devireddy

2014

Molecular and Cellular Biology

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Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.

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“…In vitro approaches have also demonstrated the potential of TLX1 to disrupt normal hematopoietic processes and promote the immortalization of murine progenitor cells derived from various hematopoietic sources, including bone marrow, fetal liver, yolk sac and embryonic stem cells [11,17-21] (reviewed in [22]). Several studies have provided evidence that TLX1 induces progenitor immortalization by blocking differentiation while concurrently increasing replicative capacity [23-27].…”

Section: Introductionmentioning

confidence: 99%

The DN2 Myeloid-T (DN2mt) Progenitor is a Target Cell for Leukemic Transformation by the TLX1 Oncogene

et al. 2013

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Introduction Inappropriate activation of the TLX1 (T-cell leukemia homeobox 1) gene by chromosomal translocation is a recurrent event in human T-cell Acute Lymphoblastic Leukemia (T-ALL). Ectopic expression of TLX1 in murine bone marrow progenitor cells using a conventional retroviral vector efficiently yields immortalized cell lines and induces T-ALL-like tumors in mice after long latency. Methods To eliminate a potential contribution of retroviral insertional mutagenesis to TLX1 immortalizing and transforming function, we incorporated the TLX1 gene into an insulated self-inactivating retroviral vector. Results Retrovirally transduced TLX1-expressing murine bone marrow progenitor cells had a growth/survival advantage and readily gave rise to immortalized cell lines. Extensive characterization of 15 newly established cell lines failed to reveal a common retroviral integration site. This comprehensive analysis greatly extends our previous study involving a limited number of cell lines, providing additional support for the view that constitutive TLX1 expression is sufficient to initiate the series of events culminating in hematopoietic progenitor cell immortalization. When TLX1-immortalized cells were co-cultured on OP9-DL1 monolayers under conditions permissive for T-cell differentiation, a latent T-lineage potential was revealed. However, the cells were unable to transit the DN2 myeloid-T (DN2mt)-DN2 T-lineage determined (DN2t) commitment step. The differentiation block coincided with failure to upregulate the zinc finger transcription factor gene Bcl11b, the human ortholog of which was shown to be a direct transcriptional target of TLX1 downregulated in the TLX1+ T-ALL cell line ALL-SIL. Other studies have described the ability of TLX1 to promote bypass of mitotic checkpoint arrest, leading to aneuploidy. We likewise found that diploid TLX1-expressing DN2mt cells treated with the mitotic inhibitor paclitaxel bypassed the mitotic checkpoint and displayed chromosomal instability. This was associated with elevated expression of TLX1 transcriptional targets involved in DNA replication and mitosis, including Ccna2 (cyclin A2), Ccnb1 (cyclin B1), Ccnb2 (cyclin B2) and Top2a (topoisomerase IIα). Notably, enforced expression of BCL11B in ALL-SIL T-ALL cells conferred resistance to the topoisomerase IIα poison etoposide. Conclusion Taken together with previous findings, the data reinforce a mechanism of TLX1 oncogenic activity linked to chromosomal instability resulting from dysregulated expression of target genes involved in mitotic processes. We speculate that repression of BCL11B expression may provide part of the explanation for the observation that aneuploid DNA content in TLX1+ leukemic T cells does not necessarily portend an unfavorable prognosis. This TLX1 hematopoietic progenitor cell immortalization/T-cell differentiation assay should help further our understanding of the mechanisms of TLX1-mediated evolution to malignancy and has the potential to be a useful predictor of disease response to novel thera...

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TLX1 (HOX11) Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors

Cited by 6 publications

References 42 publications

Hematopoietic immortalizing function of the NKL‐subclass homeobox geneTLX1

Hematopoietic immortalizing function of the NKL‐subclass homeobox geneTLX1

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

The DN2 Myeloid-T (DN2mt) Progenitor is a Target Cell for Leukemic Transformation by the TLX1 Oncogene

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